Methods and compositions to regulate iron metabolism

ABSTRACT

The present invention provides new systems and strategies for the regulation of iron metabolism in mammals. In particular, methods of using agonists and antagonists of TGF-β superfamily members to modulate the expression or activity of hepcidin, a key regulator of iron metabolism, are described. The inventive methods find applications in the treatment of diseases associated with iron overload, such as juvenile hemochromatosis and adult hemochromatosis, and in the treatment of diseases associated with iron deficiency, such as anemia of chronic disease and EPO resistant anemia in end-stage of renal disease. The present invention also relates to screening tools and methods for the development of novel drugs and therapies for treating iron metabolism disorders.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a divisional application of U.S. patentapplication Ser. No. 111/884,509, filed on Jun. 17, 2008, which is aU.S. National Stage of International Application No. PCT/US2006/005367,filed on Feb. 16, 2006, which claims priority to U.S. ProvisionalApplication No. 60/653,479 filed on Feb. 16, 2005 and entitled “Methodsand Compositions to Regulate Iron Metabolism”. The ProvisionalApplication which is incorporated herein by reference in its entiretytheir entireties.

REFERENCE TO A “SEQUENCE LISTING”

The sequence listing material in the text file entitled“11884509_SeqList.txt” (30,633 bytes), which was created on Nov. 8,2010, is herein incorporated by reference in its entirety.

BACKGROUND OF THE INVENTION

Iron is an essential element for the growth and survival of nearly allliving organisms (P. Aisen et al., J. Biochem. Cell Biol., 2001, 33:940-959) except for a few unusual bacterial species. It plays animportant role in oxygen transport and storage (in combination withoxygen-binding molecules such as hemoglobin and myoglobulin) and is akey component of many enzymes that catalyze the redox reactions requiredfor the generation of energy (e.g., cytochromes), the production ofvarious metabolic intermediates, and for host defense (e.g.,nicotinamide adenine dinucleotide phosphate [NADPH] oxidase). Iron canalso be toxic. It catalyzes the generation of reactive radical speciesthat can attack cellular membranes, proteins, and DNA (J. M. C.Gutteridge et al., Biochem. J., 1982, 296: 605-609), and activatesNF-κB, the prototype transcription factor for genes involved ininflammation (S. Xiong et al., J. Biol. Chem., 2003, 278: 17646-17654).At high levels, iron accumulation in tissues is damaging.

To prevent iron deficiency or iron overload, virtually all organismshave developed elaborate mechanisms for regulating iron intake andefflux (C. Finch, Blood, 1994, 84: 1697-1702). In adult mammals, ironhomeostasis depends upon regulated absorption of iron by the enterocyte,a highly specialized cell of the duodenum that coordinates dietary ironuptake and transport into the body. In the fetus, the mechanismsinvolved in placental materno-fetal iron transport are also tightlyregulated. Iron is stored in the body in the form of the proteincomplexes, ferritin and hemosiderin, and is transported in the plasmavia the protein complex, transferrin. Under normal circumstances, onlytrace amounts of iron exist outside these physiologic sinks, althoughstored iron can be mobilized by reuse. Perturbations in these highlyregulated mechanisms can lead to iron overload or iron deficiency in thebody.

Iron deficiency is the most common nutritional disorder in the world. Asmany as 4-5 billion people 65-80% of the world's population) may be irondeficient; and 2 billion people (over 30% of the world's population,mostly children and women of childbearing age) are anemic, mainly due toiron deficiency. In developing countries, the disease is exacerbated bymalaria and worm infections. Iron deficiency affects more people thanany other condition, constituting a public health condition of epidemicproportions. Iron overload disorders are less prevalent; however, theycan lead to serious life-threatening conditions. Worldwide, some 24million people of northern European ancestry suffer from a geneticdisorder called hemochromatosis. Another 600 million carry one of thegenes responsible for the disorder, and absorb up to 50% more iron thannon-carriers. The disease leads to iron accumulation, particularly inthe liver and other storage organs, which can cause organ failure (likecirrhosis of the liver), heart attack, cancer, and pancreatic damage.

Dysfunctions in iron metabolism pose a major problem worldwide due notonly to their frequency but also to the lack of therapeutic options (N.C. Andrews, N. Engl. J. Med., 1999, 341: 1986-1995). Iron overloadconditions are generally treated by administration of iron chelatingagents, which exert their effects by remobilizing accumulated iron andallowing for its excretion. In practice, however, none of the chelatingagents which have been evaluated to date have proved entirelysatisfactory, suffering from poor gastrointestinal absorption, andeither low efficacy, poor selectivity, or undesirable side effects. Thepreferred treatment for reducing iron levels in most hemochromatosispatients is called therapeutic phlebotomy, a procedure which simplyconsists of removing blood from the body. Patients with hemochromatosisusually need a large number of phlebotomies in a relative short periodof time (up to once or twice a week). Thus, in addition to carrying thesame risks as with any blood donation (e.g., nausea, vomiting,dizziness, fainting, hematoma, seizures or local infection), phlebotomycan also be highly constraining to the patient.

Several forms of iron salt are used to treat iron deficiency conditions.It generally takes several months of replacement therapy to replenishbody iron stores. Some patients have difficulty tolerating iron salts,because these substances tend to cause gastrointestinal distress.Studies have also reported that liquid iron-salt preparations, given toyoung children, may cause permanent staining of the teeth. However, moreproblematic is the finding that high doses of iron supplements, takenorally or by injection, can increase susceptibility to bacterialinfection.

Clearly, the development of novel agents and methods for the preventionand treatment of iron metabolism disorders, remains highly desirable.

SUMMARY OF THE INVENTION

The present invention provides improved systems and strategies forregulating iron metabolism in mammals, including humans. In particular,the invention encompasses reagents and processes for the treatment ofconditions associated with iron deficiency or iron overload. Theinvention also provides screening tools and methods for theidentification of compounds useful for the treatment of iron metabolismdisorders. Compared to existing therapies such as iron supplementation,iron chelation, and phlebotomy, the inventive methods and compositionsare less likely to induce undesirable side-effects.

In general, the present invention involves the use of modulators of thesignaling activity of members of the TGF-β superfamily to control and/orregulate the expression or activity of hepcidin, a key regulator of ironmetabolism in mammals.

More specifically, in one aspect, the present invention provides methodsfor regulating hepcidin expression or activity in a subject byadministering to the subject an effective amount of a compound thatmodulates the signaling activity of at least one TGF-β superfamilymember. The present invention also provides methods for regulatinghepcidin expression or activity in a biological system by contacting thebiological system with an effective amount of a compound that modulatesthe signaling activity of at least one TGF-β superfamily member. Incertain embodiments, the TGF-β superfamily member is TGF-β or BMP. Thecompound administered to the subject or contacted with the biologicalsystem may comprise an agent selected from the group consisting of anagonist of TGF-β, an antagonist of TGF-β, an agonist of BMP, anantagonist of BMP, or combinations thereof. The biological system may bea cell, a biological fluid, a biological tissue or an animal.

In certain embodiments, the agent is selected from the group consistingof a HJV.Fc fusion protein, a Dragon.Fc fusion protein, a DLN.Fc fusionprotein, a sTβRII.Fc fusion protein, a sTβRII-B.Fc fusion protein, and asTβRIIIΔ.Fc fusion protein. In some embodiments, the agent comprises afusion protein selected from the group consisting of a mutant HJV.Fcfusion protein, a mutant Dragon.Fc fusion protein and mutant DLN.Fcfusion protein, wherein the mutant fusion protein is non-proteolyticallycleavable.

In methods for inhibiting hepcidin expression or activity in a subjector a biological system, the compound administered to the subject orcontacted with the biological system is preferably an agonist of TGF-β,an antagonist of BMP, or combinations thereof. In methods for enhancinghepcidin expression or activity in a subject or a biological system, thecompound administered to the subject or contacted with the biologicalsystem is preferably an antagonist of TGF-β, an agonist of BMP, orcombinations thereof.

In another aspect, the present invention provides methods for regulatingiron metabolism or an iron metabolic process' in a subject or abiological system by administering to the subject or contacting thebiological system with an effective amount of a compound that modulatesthe signaling activity of at least one TGF-β superfamily member. Theiron metabolic process may be iron uptake, iron absorption, irontransport, iron storage, iron processing, iron mobilization, ironutilization, or combinations thereof.

When the subject or biological system exhibits or is at risk ofexhibiting iron deficiency, the compound used in these methods ispreferably an agonist of TGF-β, an antagonist of BMP, or combinationsthereof. When the subject or biological system exhibits or is at risk ofexhibiting iron overload, the compound is, preferably, an antagonist ofTGF-β, an agonist of BMP, or combinations thereof.

In still another aspect, the present invention provides methods fortreating or preventing conditions associated with perturbations in ironmetabolism in a subject. The inventive methods comprise administering tothe subject an effective amount of a compound that modulates thesignaling activity of at least one TGF-β superfamily member. In certainembodiments, the TGF-β superfamily member is TGF-β or BMP. In someembodiments, administration of the compound to the subject results inregulation of hepcidin expression or activity in the subject.

Compounds administered in the inventive methods may comprise an agentselected from the group consisting of an agonist of TGF-β, an antagonistof TGF-β, an agonist of BMP, an antagonist of BMP, or combinationsthereof. In certain embodiments, the agent is selected from the groupconsisting of a HJV.Fc fusion protein, a Dragon.Fc fusion protein, aDLN.Fc fusion protein, a sTβRII.Fc fusion protein, a sTβRII-B.Fc fusionprotein, and a sTβRIIIΔ.Fc fusion protein. In some embodiments, theagent comprises a fusion protein selected from the group consisting of amutant HJV.Fc fusion protein, a mutant Dragon.Fc fusion protein andmutant DLN.Fc fusion protein, wherein the mutant fusion protein isnon-proteolytically cleavable.

When the subject has or is at risk of having a condition associated withiron deficiency, the compound used in these methods is, preferably, anantagonist of BMP, an agonist of TGF-β, or combinations thereof.Conditions associated with iron deficiency that can be treated and/orprevented by methods of the present invention include, but are notlimited to, anemia of chronic disease, iron deficiency anemia,functional iron deficiency, and microcytic anemia. In certainembodiments, the methods further comprise administering an ironsupplementation treatment to the subject.

When the subject has or is at risk of having a condition associated withiron overload, the compound used in the methods of treatment is,preferably, an agonist of BMP, an antagonist of TGF-β, or combinationsthereof. Conditions associated with iron overload that can be treatedand/or prevented by methods of the present invention include, but arenot limited to, adult hemochromatosis and juvenile hemochromatosis. Incertain embodiments, the methods further comprise administering an ironchelation treatment to the subject. In some embodiments, the methodsfurther comprise performing phlebotomy to the subject.

In yet another aspect, the present invention provides methods foridentifying compounds that regulate hepcidin expression or activity in abiological system, and methods for identifying compounds that regulateiron metabolism or an iron metabolic process in a biological system. Inthese methods, the biological system preferably expresses at least oneTGF-β superfamily member.

These methods comprise incubating the biological system with a candidatecompound under conditions and for a time sufficient for the candidatecompound to modulate the signaling activity of the TGF-β superfamilymember; thereby obtaining a test system; measuring, in the test system,at least one factor that is representative of the signaling activity ofthe TGF-β superfamily member; incubating the system under the sameconditions and for the same time absent the candidate compound, therebyobtaining a control system; measuring the factor in the control system;comparing the factor measured in the test and control systems; anddetermining that the candidate compound regulates hepcidin expression oriron metabolism in the system, if the factor measured in the test systemis less than or greater than the factor measured in the control system.

In certain embodiments, the TGF-β superfamily member is TGF-β or BMP,and the compound identified as regulator is selected from the groupconsisting of an agonist of TGF-β, an antagonist of TGF-β, an agonist ofBMP, and an antagonist of BMP. In some embodiments, the agent comprisesa fusion protein selected from the group consisting of a mutant HJV.Fcfusion protein, a mutant Dragon.Fc fusion protein and mutant DLN.Fcfusion protein, wherein the mutant fusion protein is non-proteolyticallycleavable.

The present invention also provides pharmaceutical compositionscomprising a pharmaceutically acceptable carrier, and an effectiveamount of at least one regulator of iron metabolism or at least oneregulator of hepcidin expression or activity identified by the inventivescreening methods. Also provided are methods of using these identifiedregulators in the treatment or prevention of conditions associated withperturbations in iron metabolism.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 is a graph showing the effects of BMP-2, Noggin.Fc (which isknown to inhibit endogenous BMP signaling), and Fe-NTA(non-transferrin-bound iron) on the hepcidin/actin ratio in HepG2 cellscompared to the hepcidin/actin ratio in control cells, C (i.e., HepG2cells incubated in the absence of these agents).

FIG. 2 is a graph showing the effects of BMP-2, Noggin.Fc, and TGF-β1 onthe hepcidin/actin ratio in HepG2 cells compared to the hepcidin/actinratio in control cells, C (i.e., HepG2 cells incubated in the absence ofthese agents).

FIG. 3 shows a set of Western blot analyses of HJV protein in the liver(A) and transfected CHO cells (B), and of soluble HJV.Fc fusion protein(C).

FIG. 4 is a set of three graphs showing measurements of luciferaseactivity in HepG2 cells transfected with a BMP-responsive luciferasereporter (A, C) or a TGF-β responsive luciferase reporter (B) incubatedwith or without BMP-2, BMP-4 or TGF-β1.

FIG. 5 is a graph showing measurements of luciferase activity in HepG2cells transfected with BMP-responsive luciferase reporter and HJV cDNAor empty vector, and incubated with or without exogenous BMP-2 in thepresence or absence of Noggin.

FIG. 6(A) is a graph showing results of radioactivity measurements fromHJV.Fc incubated with ¹²⁵I-labeled BMP-2. FIG. 6(B) is a gel showingthat ¹²⁵I-BMP-2 can be chemically crosslinked with HJV.Fc in thepresence of DSS (bar 4) and that this crosslinking can be inhibited byexcess cold BMP-2 (bar 5).

FIG. 7 is a graph reporting radioactivity measurements from HJV.Fcincubated with ¹²⁵I-labeled BMP-2 with or without excess cold BMP-2, -4,-7, or TGF-β1.

FIG. 8 is a set of two graphs showing measurements of luciferaseactivity in HepG2 cells co-transfected with BMP-responsive luciferasereporter and HJV either alone or in combination with dominant negativeBMP type I receptor ALK3 (ALK3 DN) or ALK6 (ALK6 DN) (FIG. 8(A)), orwith wild-type (WT) versus dominant negative (DN) R-Smad 1 (FIG. 8(B)).

FIG. 9 shows two Western blot analyses of mutant HJVG313V (FIG. 9(A))and soluble HJVG313V.Fc cDNA (FIG. 9(B)) made using PCR and subcloningtechniques, and transfected into CHO cells

FIG. 10 shows immunofluorescence microscopy images of unpermeabilizedcells transfected with wild-type HJV and mutant HJVG313V.

FIG. 11 is a graph reporting measurements of luciferase activity inHepG2 cells transfected with BMP-responsive luciferase reporter alone orin combination with increasing concentrations of wild-type HJV or mutantHJVG13V cDNA.

FIG. 12 is a table reporting measurements of serum iron and total ironbinding capacity in mice after intraorbital treatment with BMP-2 ligand.

FIG. 13 is a gel showing that mouse RMGa-D169A.Fc fusion protein is notproteolytically cleaved compared to mouse RMGa.Fc fusion protein.

FIG. 14 is a gel showing that mouse Dragon-D171A.Fc fusion protein isnot proteolytically cleaved compared to mouse Dragon.Fc fusion protein.

FIG. 15 is a gel showing that human HJV-D172A is not proteolyticallycleaved compared to human HJV.

FIG. 16 shows a schematic describing how hepcidin modulates irontransport from enterocytes and macrophages. Hepcidin production ismodulated by inflammatory signals, iron levels and signals from the bonemarrow (erythroid drive). Hepcidin is initially produced as prohepcidin(84 amino acids), which is processed by cleavage to the putativelyactive form of 25 amino acids. Elevated levels of hepcidin prevent ironuptake from the intestine and iron release from macrophages.

FIG. 17 shows a table summarizing genetic analysis of kindreds affectedwith juvenile hemochromatosis. Haplotype data in ten Greek families withjuvenile hemochromatosis. Genotypes are shown for 27 informative markersfrom the 1p13-q23 genetic interval in the indicated individuals, witheach of the consensus haplotypes shaded in a different color. Markersdesignated ‘D1S’ are described in build 33 of the human genome, andnewly generated microsatellite markers are designated by repeat type andhuman genomic clone accession numbers. ND indicates genotypes that werenot determined. Alleles of uncertain phase are underlined, inferredalleles are italicized and alleles observed most frequently in 56 Greekcontrol chromosomes are shown in bold. Marker order is based on arevised interpretation of the April 2003 build 33 of the human genomeassembly. The red bar indicates the critical interval associated withjuvenile hemochromatosis.

FIG. 18A shows a schematic displaying HFE2 gene structure andbioinformatics analysis. Transcript 1 was determined from sequencing anovel RT-PCR cDNA clone from human liver RNA. Alternatively splicedtranscripts 2-5 were based on occurrence in EST or cDNA clones in publicdatabases and RT-PCR experiments. Each of the five putative transcriptsof HFE2 may be translated into a polypeptide. Transcripts 3, 4 and 5generate the same protein; hence, there are three hemojuvelin isoformsof 426, 313 or 200 amino acids. Exon 2 was predicted in Ensemb1 forhuman HFE2 based on a rat cDNA clone containing this exon (incorrectlyannotated as human but 100% identical with rat genomic sequence).Additional mouse ESTs, conservation of the exon in human genomicsequence and a novel human cDNA clone verified the coding region oftranscript 1. Untranslated sequence is colored white, translatedsequence black. Below the transcripts is shown a version of the longestopen reading frame (ORF) with protein domains parsed across the exonsand codon numbers given at splice junctions (SP, signal peptide; RGD,tri-amino acid motif; vWf, partial von Willebrandt factor; TM,transmembrane). Gray horizontal bar at bottom indicates northern-blotprobe.

FIG. 18B shows a multiple sequence alignment of HFE2 (hemojuvelin) withorthologs of mouse, rat and zebrafish and paralogs of human and chicken.The longest cDNA sequence (transcript 1) and its predicted proteinsequence were used as the basis for sequence numbering beginning fromthe putative initiating methionine. Above sequences, protein functionaldomains are shown as horizontal bars. Amino acid changes in individualswith juvenile hemochromatosis are indicated by arrows.

FIG. 19 shows a northern blot displaying tissue expression ofhemojuvelin. Northern blots of human tissues were probed with sequencesencompassing exon 4 of HFE2 and then reprobed with probes for hepcidin(HAMP) and β-actin (ACTB). The ACTB probe highlights a second isoformspecific to skeletal muscle and heart, in addition to the ubiquitoustranscript in these tissues. Sizes are relative to lane standards. PBL,peripheral blood lymphocytes.

FIG. 20 shows a schematic summarizing that anemia of inflammation andhemochromatosis represent opposite ends of the phenotypic spectrum ofiron-related disorders. Anemia of inflammation (AI) is characterized byhigh levels of hepcidin, which leads to iron deficiency and iron-richmacrophages. In contrast, in hemochromatosis, hepcidin levels are lowwith enhanced intestinal absorption and whole-body iron overload.Macrophages in hemochromatosis are iron-depleted. Juvenilehemochromatosis (JH) and adult-onset hereditary hemochromatosis (HH)both show iron overload with iron-depleted macrophages, but thephenotype is more severe in juvenile hemochromatosis.

DEFINITIONS

Throughout the specification, several terms are employed that aredefined in the following paragraphs.

As used herein, the term “a TGF-β superfamily member” refers to anymember of the TGF-β superfamily, which includes, among others, activins,inhibins, Transforming Growth Factors beta (TGF-βs), Growth andDifferentiation Factors (GDFs), Bone Morphogenetic Proteins (BMPs), andMüllerian Inhibiting Substance (MIS). In the context of the presentinvention, certain preferred TGF-β superfamily members include TGF-βsand BMPs.

The term “perturbations” when applied to iron metabolism or an ironmetabolic process, refers to any disturbances, dysregulations and/ordeviations from normal state, function and/or level of activity. Ironmetabolic processes include iron uptake, iron absorption, irontransport, iron storage, iron processing, iron mobilization, and ironutilization. Generally, perturbations in iron metabolism result in ironoverload or iron deficiency. As used herein, the term “iron overload”refers to an amount of iron present in a subject's tissue or in abiological system which is significantly above the normal level in thatparticular tissue or that particular biological system. The term “irondeficiency” refers to an amount of iron present in a subject's tissue orin a biological system which is significantly below the normal level inthat particular tissue or that particular biological system. An amountof iron significantly below or significantly above the normal levelcorresponds to any amount of iron that is physiologically undesirableand/or that is or may become harmful to the subject or the biologicalsystem. Methods for the determination of iron levels are known in theart (see below).

As used herein, the term “condition associated with perturbations ofiron metabolism or an iron metabolic process” refers to any disease,disorder, syndrome or condition that is characterized by iron overloador iron deficiency.

The term “prevention” is used herein to characterize a method that isaimed at delaying or preventing the onset of a pathophysiologicalcondition associated with perturbations in iron metabolism (for examplein a subject which may be predisposed to the condition but has not yetbeen diagnosed as having it).

The term “treatment” is used herein to characterize a method that isaimed at (1) delaying or preventing the onset of a condition associatedwith perturbations in iron metabolism; or (2) slowing down or stoppingthe progression, aggravation, or deterioration of the symptoms of thecondition; or (3) bringing about ameliorations of the symptoms of thecondition; or (4) curing the condition. A treatment may be administeredprior to the onset of the disease, for a prophylactic or preventiveaction. It may also be administered after initiation of the disease, fora therapeutic action.

The terms “compound” and “agent” are used herein interchangeably. Theyrefer to any naturally occurring or non-naturally occurring (i.e.,synthetic or recombinant) molecule, such as a biological macromolecule(e.g., nucleic acid, polypeptide or protein), organic or inorganicmolecule, or an extract made from biological materials such as bacteria,plants, fungi, or animal (particularly mammalian, including human) cellsor tissues. The compound may be a single molecule, a mixture of two ormore molecules, or a complex of at least two molecules.

The term “candidate compound” refers to a compound or agent (as definedabove) that is to be tested for an activity of interest. In certainscreening methods of the invention, candidate compounds are evaluatedfor their ability to regulate hepcidin expression and/or to regulateiron metabolism through modulation of the signaling activity of a TGF-βsuperfamily member.

The term “regulation” when applied to a biological phenomenon (such asiron metabolism, and hepcidin expression or activity) refers to aprocess that allows for control of the biological phenomenon. The term“regulation” also refers to the ability of a compound to control thebiological phenomenon. For example, a process or a compound thatregulates iron metabolism has the ability to decrease iron levels in asubject or biological system that exhibits iron overload; and/or theability to increase iron levels in a subject or biological system thatexhibits iron deficiency. In the context of the present invention, themechanism by which regulation of the biological phenomenon takes placeis preferably through modulation of the signaling activity of a TGF-βsuperfamily member. In the screening methods of the invention, when acandidate compound is found to regulate hepcidin expression or activity,it is identified as a “regulator” of the expression or activity ofhepcidin.

As used herein, the term “modulation of the signaling activity of aTGF-β superfamily member” refers to the ability of a compound toincrease or prolong, or to decrease or reduce the duration of the effectof a TGF-β superfamily member. In the screening methods of theinvention, when a candidate compound is found to induce such anenhancement or inhibition, it is identified as a “modulator” of thesignaling activity of the TGF-β superfamily member.

The term “agonist” is intended to be used as is accepted in the art. Ingeneral, the term refers to a compound that increases or prolongs theduration of the effect of a polypeptide or a nucleic acid. An agonistmay be a direct agonist, in which case it is a molecule that exerts itseffect by interacting with (e.g., binding to) the polypeptide or nucleicacid, or an indirect agonist, in which case it exerts its effect via amechanism other than by interaction with the polypeptide or nucleic acid(e.g., by altering the expression or stability of the polypeptide ornucleic acid, by altering the expression or activity of a target of thepolypeptide or nucleic acid, by interacting with an intermediate in apathway involving the polypeptide or nucleic acid, etc.).

The term “antagonist” is intended to be used as is accepted in the art.In general, the term refers to a compound that decreases or reduces theduration of the effect of a polypeptide or a nucleic acid. An antagonistmay be a direct antagonist, in which case it is a molecule that exertsits effect by interacting with (e.g., binding to) the polypeptide ornucleic acid, or an indirect antagonist, in which case it exerts itseffect via a mechanism other than by interaction with the polypeptide ornucleic acid (e.g., by altering the expression or stability of thepolypeptide or nucleic acid, by altering the expression or activity of atarget of the polypeptide or nucleic acid, by interacting with anintermediate in a pathway involving the polypeptide or nucleic acid,etc.).

As used herein, the term “effective amount” refers to any amount of acompound or agent that is sufficient to fulfill its intended purpose(s).In the context of the present invention, the purpose(s) may be, forexample: to modulate the signaling activity of a TGF-β superfamilymember; and/or to regulate hepcidin expression or activity; and/or toregulate iron metabolism or an iron metabolic process; and/or to delayor prevent the onset of a condition associated with perturbations iniron metabolism; and/or to slow down or stop the progression,aggravation, or deterioration of the symptoms of the condition; and/orto bring about ameliorations of the symptoms of the condition; and/or tocure the condition.

The term “subject” refers to a human or another mammal, that can beaffected by a pathophysiological condition associated with perturbationsin iron metabolism but may or may not have such a condition.

The terms “system” and “biological system” are used hereininterchangeably. A system may be any biological entity that can exhibitiron overload or iron deficiency. The biological system is preferablyone that expresses at least one TGF-β superfamily member. Some preferredsystems express TGF-β and/or BMP. The biological system may alsopreferably express hepcidin or comprise hepcidin. In the context of thisinvention, in vitro, in vivo, and ex vivo systems are considered; andthe system may be a cell, a biological fluid, a biological tissue, or ananimal. A system may, for example, originate from a live subject (e.g.,it may be obtained by drawing blood, or by biopsy), or from a deceasedsubject (e.g., it may be obtained at autopsy).

As used herein, the term “biological fluid” refers to a fluid producedby and obtained from a subject. Examples of biological fluids include,but are not limited to, urine, blood serum, and plasma. In the presentinvention, biological fluids include whole or any fraction of suchfluids derived by purification, for example, by ultra-filtration orchromatography. As used herein, the term “biological tissue” refers to atissue obtained from a subject. The biological tissue may be whole orpart of any organ or system in the body (e.g., liver, gastrointestinaltract, kidney, pancreas, and the like).

A “pharmaceutical composition” is defined herein as comprising at leastone compound of the invention (i.e., a candidate compound identified byan inventive screening method as a regulator of iron metabolism, and/ora regulator of hepcidin expression or activity), and at least onepharmaceutically acceptable carrier.

As used herein, the term “pharmaceutically acceptable carrier” refers toa carrier medium which does not interfere with the effectiveness of thebiological activity of the active ingredients and which is notexcessively toxic to the hosts at the concentrations at which it isadministered. The term includes solvents, dispersion media, coatings,antibacterial and antifungal agents, isotonic agents, absorptiondelaying agents, and the like. The use of such media and agents for theformulation of pharmaceutically active substances is well known in theart (see, for example, Remington's Pharmaceutical Sciences, E. W.Martin, 18^(th) Ed., 1990, Mack Publishing Co., Easton, Pa.).

DETAILED DESCRIPTION OF CERTAIN PREFERRED EMBODIMENTS

As mentioned above, the present invention provides improved systems andstrategies for regulating iron metabolism in mammals. In particular, theinventive compounds and methods are less likely than existing therapiesto induce undesirable side effects.

I. Agonists and Antagonists of TGF-β Superfamily Members as Regulatorsof Iron Metabolism

The present invention encompasses the discovery that certain members ofthe TGF-β superfamily can regulate the expression of hepcidin, a keyregulator of iron metabolism in mammals. As described in Example 1, thepresent Applicants have recognized that BMP (bone morphogenetic protein)signaling induces hepcidin expression in HepG2 liver hepatoma cells,while TGF-β (Transforming Growth Factor-beta) signaling inhibits theexpression of hepcidin. Furthermore, using Noggin, a well-known BMPantagonist, the Applicants have shown that inhibition of BMP signalingresulted in reduction of hepcidin expression.

Accordingly, the present invention provides methods of using agonistsand/or antagonists of members of the TGF-β superfamily to regulatehepcidin expression or activity, which, in turn, regulates ironmetabolism.

A—Hepcidin

Hepcidin is a small, cysteine-rich cationic peptide with antimicrobialproperties that was purified only recently from human urine and plasmaultra-filtrate (C. H. Park et al., J. Biol. Chem., 2001, 276: 7806-7810;A. Krause et al., FEBS Lett., 2000, 480: 147-150). This peptide of 20,22 or 25 amino acids, differing by amino acid terminal truncation, formsa short hairpin with two arms linked by four disulfide bridges in aladder-like fashion. Hepcidin contains eight cysteine residues that areconserved among species (G. Nicolas et al., Proc. Natl. Acad. Sci. USA,2001, 98: 878-885; C. Pigeon et al., J. Biol. Chem., 2001, 276:7811-7819). Even though the peptide was first isolated from urine andblood, hepcidin is predominantly expressed in the liver in both mice andhumans. Expression is also detectable in the heart and brain, but to amuch less extent (C. H. Park et al., 2001; C. Pigeon et al., 2001).Recently, hepcidin has also been found to be expressed in the kidney (H.Kulaksiz et al., J. Endocrinol., 2005, 184: 361-370).

Only one copy of the gene exists in humans, whereas two hepcidin genes(Hepc 1 and Hepc 2) have been reported in mice (G. Nicolas et al., Proc.Natl. Acad. Sci. USA, 2001, 98: 878-885; C. Pigeon et al., J. Biol.Chem., 2001, 276: 7811-7819). Both the human and mouse hepcidin genesconsist of three exons and two introns, with the third exon encoding themature peptide found in urine. In humans and rats, the exons encode an84 (83 in mice) amino acid precursor, including a putative 24 amino acidsignal peptide.

The connection between hepcidin and iron metabolism was first made byPigeon et al. (J. Biol. Chem., 2001, 276: 7811-7819) while investigatinghepatic responses to iron overload. Other studies have shown that micelacking hepcidin mRNA developed iron overload affecting liver andpancreas, with deficit in the macrophage-rich spleen (G. Nicolas et al.,Proc. Natl. Acad. Sci. USA, 2001, 98: 8780-8785). Transgenic miceoverexpressing hepcidin were observed to die at birth of severe irondeficiency (G. Nicolas et al., Proc. Natl. Acad. Sci. USA, 2002, 99:4596-4601). These studies suggested that hepcidin inhibits ironabsorption in the small intestine, the release of recycled iron frommacrophages (R. E. Fleming and W. S. Sly, Proc. Natl. Acad. Sci. USA,2001, 98: 8160-8162), and transport of iron across the placenta (G.Nicolas et al., Proc. Natl. Acad. Sci. USA, 2002, 99: 4596-4601). Inagreement with animal studies, patients with large hepatic adenomas andotherwise unexplained iron refractory anemia exhibit overexpressedhepcidin mRNA in the liver (D. A. Weinstein et al.; Blood, 2002, 100:3776-3781). Recent studies have found abnormal hepcidin expression anddisrupted hepcidin regulation (K. R. Bridle et al., Lancet, 2003, 361:669-673; H. Kulaksiz et al., Gut, 2004, 53: 735-743) in hemochromatosisgene (HFE)-associated hemochromatosis and association of hepcidinmutations with severe juvenile hemochromatosis (A. Roetto et al., NatureGenetics, 2003, 33: 21-22). Based on these and other observations, ithas been suggested that hepcidin is a key component of iron homeostasisthat acts as a negative regulator of iron metabolism.

B—TGF-β Superfamily Members

The TGF-β superfamily of ligands presently comprises more than 30members, including, among others, activins, inhibins, TransformingGrowth Factors-beta (TGF-βs), Growth and Differentiation Factors (GDFs),Bone Morphogenetic Proteins (BMPs), and Müllerian inhibiting Substance(MIS). All of these molecules are peptide growth factors that arestructurally related to TGF-β. They all share a common motif called acysteine knot, which is constituted by seven especially conservativecysteine residues organized in a rigid structure (J. Massagué, Annu.Rev. Biochem., 1998, 67: 753-791). Unlike classical hormones, members ofthe TGF-β superfamily are multifunctional proteins whose effects dependon the type and stage of the target cells as much as the growth factorsthemselves.

TGF-β superfamily members suitable for use in the practice of themethods of the present invention include any member of the TGF-βsuperfamily whose signaling activity can regulate the expression oractivity of hepcidin. Preferred TGF-β superfamily members include, butare not limited to, TGF-βs and BMPs.

Transforming Growth Factors-Beta (TGF-βs)

In certain embodiments of the invention, the TGF-β superfamily member isTGF-β. TGF-βs are extracellular polypeptides that are implicated in abroad range of biological processes (J. Massagué, Ann. Rev. Cell. Biol.,1990, 6: 597-641) and play a central role in key events duringembryogenesis, adult tissue repair, and immunosuppression (M. B. Spornand A. B. Roberts, J. Cell. Biol. 1992, 119: 1017-1021; S. W. Wahl, J.Clin. Immunol. 1992, 12: 61-74; D. M. Kingsley, Genes Dev. 1994, 8:133-146). In mammals, TGF-β is produced by almost all cells of theorganism, and almost all cells can serve as targets for its effects.TGF-β is a potent regulator of cell proliferation, cell differentiation,apoptosis, and extracellular matrix production.

Mammalian cells can produce three different isoforms of TGF-β: TGF-β1,TGF-β2, and TGF-β3. These isoforms exhibit the same basic structure(they are homodimers of 112 amino acids that are stabilized by intra-and inter-chain disulfide bonds) and their amino acid sequences presenta high degree of homology (>70%). However, each isoform is encoded by adistinct gene, and each is expressed in both a tissue-specific anddevelopmentally regulated fashion (J. Massagué, Annu. Rev. Biochem.1998, 67: 753-791). According to modern concepts, TGF-β exerts itseffects by first binding to membrane receptors on the target cell,thereby initiating downstream signaling events. Cross-linking studieshave shown that TGF-β mainly binds to three high-affinity cell-surfaceproteins, called TGF-β receptors of type I, type II, and type III (J.Massagué and B. Like, J. Biol. Chem. 1985, 260: 2636-2645; S. Cheifetzet al., J. Biol. Chem. 1986, 261: 9972-9978).

Regulation of iron metabolism according to methods of the presentinvention may be achieved by inhibition or enhancement of the signalingactivity of any one of the isoforms of TGF-β (i.e., TGF-β1, TGF-β2, andTGF-β3) as long as this inhibition or enhancement results in regulationof hepcidin expression or activity.

Bone Morphogenetic Proteins (BMPs)

In other embodiments of the present invention, the TGF-β superfamilymember is BMP. BMPs were originally identified as proteins that inducebone formation at ectopic (i.e., non-skeletal) sites (A. H. Reddi, Curr.Opin. Genet. Dev., 1994, 4: 737-744). However, it is now clear that inaddition to their roles in bone and cartilage morphogenesis, BMPs arealso involved in prenatal development and postnatal growth and/or repairof the eye, heart, blood, lung, kidney, muscle, skin, and other tissues(K. A. Waite and C. Eng, Nat. Rev. Genet., 2003, 4: 763-773). Studieshave shown that BMPs play an important role in regulating proliferation,apoptosis, differentiation, and chemotaxis of various cell types,including mesenchymal cells, epithelial cells, hematopoietic cells andneuronal cells. (J. Massagué and Y. G. Chen, Genes Dev., 2000, 14:627-644; K. Miyazono et al., J. Cell Physiol., 2001, 187: 265-276: N.Morrell et al., Circulation, 2001, 104: 790-795; A. von Budnoff and K.W. Y. Cho, Dev. Biol., 2001, 239: 1-14).

In a manner similar to other members of the TGF-β superfamily, BMPsmediate their effects by forming a complex of two different types oftransmembrane serine/threonine kinase receptors: type I and type II (C.H. Heldin et al., Nature, 1997, 390: 465-471; J. Newman et al., N. Engl.J. Med., 2001, 345: 319-324; B. L. Rosenzweig et al., Proc. Natl. Acad.Sci. USA, 1995, 92: 7632-7636). Three different BMP type I receptors(activin receptor-like kinase ALK2, ALK3, and ALK6) and three BMP typeII receptors (BMP type II receptor (BMPRII); Activin type IIA receptor(ActRIIA); and Activin type BB receptor (ActRIIB)) have been identified(L. Attisano and J. L. Wrana, Science, 2002, 296: 1646-1647). BMPbinding induces phosphorylating of the type I receptor by the type IIreceptor, which leads to phosphorylation of cytoplasmicreceptor-activated Smads (C. H. Heldin et al., Nature, 1997, 390:465-471).

To date, nearly 20 BMP isoforms have been identified and characterizedin mammals and newer ones are being discovered (M. Kawabata et al.,Cytokine Growth Factor Rev., 1998, 9: 49-61). The BMP family membershave been classified in subgroups according to how closely they arerelated to each other structurally (T. Sakou, Bone, 1998, 22: 591-603;R. G. Schaub and J. Wozney, Curr. Opin. Biotechnol., 1991, 2: 868-871;J. M. Schmitt et al., J. Orthop. Res., 1999, 17: 269-278). In vivo, theBMP isoforms have different profiles of expression, different affinitiesfor receptors and therefore unique biological activities.

Regulation of iron metabolism according to methods of the presentinvention may be achieved by inhibition or enhancement of the signalingactivity of any one of the isoforms of BMP as long as this inhibition orenhancement results in regulation of hepcidin expression or activity.

C—Agonists and Antagonists of TGF-β Superfamily Members

Agonists and antagonists of a TGF-β superfamily member suitable for usein the methods of the present invention include any compound or agentthat has the ability to modulate (i.e., enhance or inhibit) thesignaling activity of the TGF-β superfamily member such that thismodulation results in regulation of hepcidin expression or activity.

Suitable agonists and antagonists include naturally-occurring agonistsand antagonists of the TGF-β superfamily member (including fragments andvariants thereof that retain the biological characteristics of thenaturally-occurring agonist and antagonist ligands). Suitable agonistsand antagonists also include synthetic or human recombinant compounds.Classes of molecules that can function as agonists include, but are notlimited to, small molecules, antibodies (including fragments or variantsthereof, such as Fab fragments, Fab′2 fragments and scFvs), andpeptidomimetics. Classes of molecules that can function as antagonistsinclude, but are not limited to, small molecules, antibodies (includingfragments or variants thereof), fusion proteins, antisensepolynucleotides, ribozymes, small interfering RNAs (sRNAi), andpeptidomimetics.

As will be appreciated by those skilled in the art, any compound oragent that is identified, for example, by the inventive screening assays(described below), as a modulator of a TGF-β superfamily member issuitable for use in the practice of methods of the present invention. Inparticular, small molecules modulators that exhibit high specificity maybe of value in these methods.

Agonists and Antagonists of BMPs

Various antagonists of BMPs are known in the art (see, for example, G.J. Thomsen et al., Trends Genet., 1997, 13: 209-211; E. Canalis et al.,Endocr. Rev. 2003, 24: 218-235; V. A. Botchkarev, J. Invest. Dermatol.,2003, 120: 36-47; U.S. Pat. No. 6,432,410, each of which is incorporatedherein by reference in its entirety). In particular, the effects of BMPscan be modulated by a group of secreted polypeptides that prevent BMPsignaling by binding BMPs, thereby precluding their binding to specificcell surface receptors. BMP antagonists suitable for use in the practiceof the present invention include, but are not limited to, Noggin,chordin, ventroptin, follistatin and follistatin-related gene (FLRG).Other suitable BMP antagonists include cerberus, gremlin, caronte, DAN,Dante, and sclerostin and other structurally related proteins, which arecollectively termed the DAN family (D. Hsu et al., Mol. Cell, 1998, 1:673-683, which is incorporated herein by reference in its entirety).Proteins of the DAN family have a conserved cysteine-knot motif, whichis also found in other growth factors, including TGF-β-like factors (J.J. Pearce et al., Dev. Biol., 1999, 209: 98-110; C. R. Rodrigez Estebanet al., Nature, 1999, 401: 243-251). However, other BMP antagonists lacksequence similarity with each other. In vivo, these BMP antagonists havedistinct expression profiles, different affinities for various BMPisoforms, and regulate different biological responses.

The present invention also provides other BMP antagonists. As reportedin Example 2, hemojuvelin (HJV) is a member of the repulsive guidancemolecule (RGM) family of proteins. Individuals with HJV mutations areknown to exhibit depressed levels of hepcidin. The present Applicantshave shown that HJV enhances BMP but not TGF-β signaling. The resultsthey obtained demonstrate that HJV binds directly to BMP-2; and that theenhancing effect of HJV on BMP signaling is reduced by administration ofNoggin, indicating that HJV's action is ligand-dependent. Accordingly, afamily of soluble HJV.Fc fusion proteins is provided herein as BMPantagonists suitable for use in the practice of the present invention.

The present Applicants have recently reported (T. A. Samad et al.,“DRAGON: a bone morphogenetic protein co-receptor”, J. Biol. Chem.,2005, 280: 14122-14129, which is incorporated herein by reference in itsentirety) that DRAGON, a 436 amino acid glycosylphosphatidylinositol(GPI)-anchored member of the RGM family, which is expressed early in thedeveloping nervous system, enhances BMP but not TGF-β signaling and actsas a BMP co-receptor. Accordingly, the present invention provides afamily of soluble DRAGON.Fc fusion proteins as BMP antagonists suitablefor use in the inventive methods. An example of a DRAGON.Fc fusionprotein that can be used in the practice of the present invention hasbeen described by the present Applicants (T. A. Samad et al., “DRAGON: Amember of the repulsive guidance molecule-related family of neuronal-and muscle-expressed membrane proteins is regulated by DRG11 and hasneuronal adhesive properties”, J. Neuroscience, 2004, 24: 2027-2036,which is incorporated herein by reference in its entirety). SolubleDRAGON.Fc fusion protein has been found to bind selectively to BMP-2 andBMP-4, but not to BMP-7 or other members of the TGF-β superfamily ofligands (T. A. Samad et al., J. Biol. Chem., 2005, 280: 14122-14129).

Also provided herein is a family of RGMa.Fc (or DLN.Fc) fusion proteinsas BMP antagonists suitable for use in the practice of the presentinvention. Like DRAGON, RGMa is a member of the repulsive guidancemolecule (RGM) family of genes. RGMa and DRAGON are expressed in acomplementary manner in the central nervous systems, where RGMa mediatesrepulsive axonal guidance and neural tube closure, while DRAGONcontributes to neuronal cell adhesion through homophilic interactions.The present Applicants have shown that RGMa enhances BMP, but not TGF-β,signals in a ligand-dependent manner in cell culture and that thesoluble extracellular domain of RGMa fused to human Fc (RGMa.Fc orDLN.Fc) forms a complex with BMP type 1 receptors and binds directly andselectively to radiolabeled BMP-2 and BMP-4 (J. L. Babitt et al.,“Repulsive guidance molecule (RGMa), a DRAGON homologue, is a bonemorphogenetic protein co-receptor”, J. Biol. Chem., 2005, 280:29820-29827, which is incorporated herein by reference in its entirety)

The present invention also provides mutant HJV, RGMa and DRAGON fusionproteins. In particular, mutant HJV, RGMa and DRAGON fusion proteins areprovided that are more stable to proteolytic cleavage than thecorresponding wild-type versions. It is known in the art that HJV, RMGaand DRAGON share a consensus proteolytic cleavage site. For human HJV,the cleavage site is situated after aspartic acid residue 172 (G.Papanikolaou et al., Nature Genetics, 2004, 36: 77-82, which isincorporated herein by reference in its entirety); for human DRAGON,after aspartic acid residue 168 (T A. Samad et al., J, Neuroscience, 24:2027-2036, which is incorporated herein by reference in its entirety);and for human RGMa, after aspartic acid residue 168 (Genbank Sequence#NM 020211).

Mutant HJV, RGMa and DRAGON fusion proteins of the present inventioncontain one mutation or more than mutation that confers stability to thefusion protein, in particular stability to proteolytic cleavage. Forexample, the aspartic acid residue situated close to the cleavage sitemay be substituted by a different residue or deleted. Alternatively oradditionally, a residue in the vicinity of the cleavage site may besubstituted by a different residue or deleted. Methods that allowspecific mutations or mutations in specific portions of a polynucleotidesequence that encodes an isolated polypeptide to provide variants areknown in the art. The present Applicants have demonstrated thefeasibility of producing mutant HJV, RGMa, and DRAGON proteins that arenot proteolytically cleaved, as reported in Example 4.

Agonists and Antagonists of TGF-βs

Multiple naturally-occurring modulators have been identified thatenhance or inhibit TGF-β signaling. Access of TGF-β ligands to receptorsis inhibited by the soluble proteins LAP, decorin and a2-macroglobulinthat bind and sequester the ligands (W. Balemans and W. Van Hul, Dev.Biol., 2002, 250: 231-250). TGF-β ligand access to receptors is alsocontrolled by membrane-bound receptors. BAMBI acts as a decoy receptor,competing with the type I receptor (D. Onichtchouk et al., Nature, 1999,401: 480-485); betaglycan (TGF-β type II receptor) enhances TGF-βbinding to the type II receptor (C. B. Brown et al., Science, 1999, 283:2080-2082; J. Massagué, Annu. Rev. Biochem., 1998, 67: 753-791; E. delRe et al., J. Biol. Chem., 2004, 279: 22765-22772); and endoglinenhances TGF-β binding to ALK1 in endothelial cells (D. A. Marchuk,Curr. Opin. Hematol., 1998, 5: 332-338; J. Massagué, Nat. Rev. Mol. CellBiol., 200, 1: 169-178; Y. Shi and J. Massagué, Cell, 2003, 113:685-700). Cripto, an EGF-CFC GPI-anchored membrane protein, acts as aco-receptor, increasing the binding of the TGF-β ligands, nodal, Vg1,and GDF1 to activin receptors (S. K. Cheng et al., Genes Dev., 2003, 17:31-36; M. M. Shen and A. F. Schier, Trends Genet., 2000, 16: 303-309)while blocking activin signaling.

Thus, agonists and antagonists of TGF-β signaling suitable for use inthe practice of the methods of the present invention includenaturally-occurring TGF-β antagonists (e.g., decorin, see, for example,Y. Yamaguchi et al., Nature, 1990, 346: 281-284, which is incorporatedherein by reference in its entirety); soluble forms ofnaturally-occurring TGF-β agonists (e.g., a soluble form of endoglin,see, for example, U.S. Pat. Nos. 5,719,120; 5,830,847; and 6,015,693,each of which is incorporated herein by reference in its entirety); aswell as inhibitors of naturally-occurring TGF-β antagonists.

Other suitable TGF-β antagonists include antagonists that have beendeveloped to suppress undesired effects of TGF-βs for therapeuticpurposes. For example, anti-TGF-β antibodies, whose dissociationconstants have been reported to be in the nanomolar range have beendescribed (U.S. Pat. No. 5,571,714, which is incorporated herein byreference in its entirety). These anti-TGF-β antibodies have beensuccessfully administered to animals with diverse pathologicalconditions (W. A. Broder et al., Nature, 1990, 346: 371-374; S. W. Wahl,J. Clin. Immunol. 1992, 12: 61-74; M. Shah et al., Lancet, 1992, 339:213-214; M. S. Steiner and E. R. Barrack, Mol. Endocrinol. 1992, 6:15-25; F. N. Ziyadeh et al., Proc. Natl. Acad. Sci. USA, 2000, 97:8015-8020).

Other TGF-β inhibitors have been developed based on an in vitro study,which showed that adenovirus-mediated transfer of a truncated TGF-β typeII receptor completely and specifically abolishes diverse TGF-βsignaling (H. Yamamoto et al., J. Biol. Chem. 1996, 271: 16253-16259,which is incorporated herein by reference in its entirety). Several ofthese truncated receptors possess potent antagonistic activity againsttheir ligands by acting as dominant-negative mutants (A. Bandyopadhyayet al., Cancer Res. 1999, 59: 5041-5046; Z. Qi et al., Proc. Natl. Acad.Sci. USA, 1999, 96: 2345-2349; T. Nakamura et al., Hepatol. 2000, 32:247-255, each of which is incorporated herein by reference in itsentirety).

Soluble forms of TGF-β type II receptor (Sakamoto et al., Gene Ther.2000, 7: 1915-1924; H. Ueno et al., Gene Ther. 2000, 11: 33-42; J.George et al., Proc. Natl. Acad. Sci. USA, 1999, 96: 12719-12724, eachof which is incorporated herein by reference in its entirety) and typeIII receptor (PCT application No. PCT/US2004/014175 to the presentApplicants, which is incorporated herein by reference in its entirety)have also been produced as fusion proteins and have successfully beenused to prevent or treat TGF-β-related pathophysiological conditions inanimal models.

II. Identification of Regulators of Iron Metabolism

In another aspect, the present invention provides methods for theidentification of compounds that regulate iron metabolism by modulatingthe signaling activity of a TGF-β superfamily member. The presentinvention also provides methods for the identification of compounds thatregulate hepcidin expression or activity by modulating the signalingactivity of a TGF-β superfamily member.

Preferably, these methods comprise incubating a biological system, whichexpresses at least one TGF-β superfamily member, with a candidatecompound under conditions and for a time sufficient for the candidatecompound to modulate the signaling activity of the TGF-β superfamilymember, thereby obtaining a test system; incubating the biologicalsystem under the same conditions and for the same time absent thecandidate compound, thereby obtaining a control system; measuring, inthe test system, at least one factor that is representative of thesignaling activity of the TGF-β superfamily member, measuring the factorin the control system; comparing the factor measured in the test andcontrol systems; and determining that the candidate compound regulateshepcidin expression (and/or regulates iron metabolism), if the factormeasured in the test system is less than or greater than the factormeasured in the control system.

The screening methods provided herein will lead to the discovery anddevelopment of regulators of iron metabolism and regulators of hepcidinexpression or activity that exert their effects by modulating thesignaling activity of one or more TGF-β superfamily members. Theseregulators may be potentially useful in the treatment of conditionsassociated with perturbations in iron metabolism.

A—Biological Systems

The assay and screening methods of the present invention may be carriedout using any type of biological systems, i.e., a cell, a biologicalfluid, a biological tissue, or an animal. In certain embodiments, thesystem is a biological entity that can exhibit iron deficiency or ironoverload (e.g., an animal model, a blood sample, or whole or part of anorgan, e.g., the liver); and/or a biological entity that expresses atleast one TGF-β family member (e.g., a cell); and/or a biological entitythat expresses hepcidin (e.g., a hepatocyte) or comprises hepcidin(e.g., a blood or urine sample).

In certain embodiments, the assay and screening methods of the presentinvention are carried out using cells that can be grown in standardtissue culture plastic ware. Such cells include all normal andtransformed cells derived from any recognized sources. Preferably, cellsare of mammalian (human or animal, such as rodent or simian) origin.More preferably, cells are of human origin. Mammalian cells may be ofany organ or tissue origin (e.g., brain, liver, blood, or kidney) and ofany cell types. Suitable cell type include, but are not limited to,epithelial cells, platelets, lymphocytes, monocytes, myocytes,macrophages, hepatocytes, cardiomyocytes, endothelial cells, tumorcells, and the like.

Cells to be used in the practice of the assays and screening methods ofthe present invention may be primary cells, secondary cells, orimmortalized cells (e.g., established cell lines). They may be preparedby techniques well known in the art (for example, cells may be obtainedby drawing blood from a patient or a healthy donor) or purchased fromimmunological and microbiological commercial resources (for example,from the American Type Culture Collection, Manassas, Va.). Alternativelyor additionally, cells may be genetically engineered to contain, forexample, a gene of interest such as a gene expressing a growth factor ora receptor.

Selection of a particular cell type and/or cell line to perform an assayaccording to the present invention will be governed by several factorssuch as the nature of the TGF-β superfamily member whose signalingactivity is to be modulated and the intended purpose of the assay. Forexample, an assay developed for primary drug screening (i.e., firstround(s) of screening) may preferably be performed using establishedcell lines, which are commercially available and usually relatively easyto grow, while an assay to be used later in the drug development processmay preferably be performed using primary or secondary cells, which areoften more difficult to obtain, maintain, and/or to grow thanimmortalized cells but which represent better experimental models for invivo situations.

Examples of established cell lines that can be used in the practice ofthe assays and screening methods of the present invention include HepG2liver hepatoma cells, Hep3B liver hepatoma cells, primary hepatocytes,and immortalized hepatocytes. Primary and secondary cells that can beused in the inventive screening methods, include, but are not limitedto, epithelial cells, platelets, lymphocytes, monocytes, myocytes,macrophages, hepatocytes, cardiomyocytes, endothelial cells, and tumorcells.

Cells to be used in the inventive assays may be cultured according tostandard cell culture techniques. For example, cells are often grown ina suitable vessel in a sterile environment at 37° C. in an incubatorcontaining a humidified 95% air-5% CO₂ atmosphere. Vessels may containstirred or stationary cultures. Various cell culture media may be usedincluding media containing undefined biological fluids such as fetalcalf serum. Cell culture techniques are well known in the art andestablished protocols are available for the culture of diverse celltypes (see, for example, R. I. Freshney, “Culture of Animal Cells: AManual of Basic Technique”, 2nd Edition, 1987, Alan R. Liss, Inc.).

In certain embodiments, the screening methods are performed using cellscontained in a plurality of wells of a multi-well assay plate. Suchassay plates are commercially available, for example, from StratageneCorp. (La Jolla, Calif.) and Corning Inc. (Acton, Mass.) and include,for example, 48-well, 96-well, 384-well and 1536-well plates.

B—Candidate Compounds

As will be appreciated by those of ordinary skill in the art, any kindof compounds or agents can be tested using the inventive methods. Acandidate compound may be a synthetic or natural compound; it may be asingle molecule, or a mixture or complex of different molecules. Incertain embodiments, the inventive methods are used for testing one ormore compounds. In other embodiments, the inventive methods are used forscreening collections or libraries of compounds. As used herein, theterm “collection” refers to any set of compounds, molecules or agents,while the term “library” refers to any set of compounds, molecules oragents that are structural analogs.

Traditional approaches to the identification and characterization of newand useful drug candidates generally include the generation of largecollections and/or libraries of compounds followed by testing againstknown or unknown targets (see, for example, WO 94/24314; WO 95/12608; M.A. Gallop et al., J. Med. Chem. 1994, 37: 1233-1251; and E. M. Gordon etal., J. Med. Chem. 1994, 37: 1385-1401). Both natural products andchemical compounds may be tested by the methods of the invention.Natural product collections are generally derived from microorganisms,animals, plants, or marine organisms; they include polyketides;non-ribosomal peptides, and/or variants thereof (for a review, see, forexample, D. E. Cane et al., Science, 1998, 82: 63-68). Chemicallibraries often consist of structural analogs of known compounds orcompounds that are identified as hits or leads via natural productscreening. Chemical libraries are relatively easy to prepare bytraditional automated synthesis, PCR, cloning or proprietary syntheticmethods (see, for example, S. H. DeWitt et al., Proc. Natl. Acad, Sci.U.S.A. 1993, 90:6909-6913; R. N. Zuckennann et al., J. Med. Chem. 1994,37: 2678-2685; Carell et al., Angew. Chem, Int. Ed. Engl. 1994, 33:2059-2060; P. L. Myers, Curr. Opin. Biotechnol. 1997, 8: 701-707).

Collections of natural compounds in the form of bacterial, fungal, plantand animal extracts are available from, for example, Pan Laboratories(Bothell, Wash.) or MycoSearch (Durham, N.C.). Libraries of candidatecompounds that can be screened using the methods of the presentinvention may be either prepared or purchased from a number ofcompanies. Synthetic compound libraries are commercially available from,for example, Comgenex (Princeton, N.J.), Brandon Associates (Merrimack,N.H.), Microsource (New Milford, Conn.), and Aldrich (Milwaukee, Wis.).Libraries of candidate compounds have also been developed by and arecommercially available from large chemical companies, including, forexample, Merck, Glaxo Welcome, Bristol-Meyers-Squibb, Novartis,Monsanto/Searle, and Pharmacia UpJohn. Additionally, naturalcollections, synthetically produced libraries and compounds are readilymodified through conventional chemical, physical, and biochemical means.

Useful regulators of iron metabolism and of hepcidin expression may befound within numerous classes of chemicals, including heterocycles,peptides, saccharides, steroids, and the like. In certain embodiments,the screening methods of the invention are used for identifyingcompounds or agents that are small molecules (i.e., compounds or agentswith a molecular weight<600-700).

The screening of libraries according to the inventive methods willprovide “hits” or “leads”, i.e., compounds that possess a desired butnot-optimized biological activity. The next step in the development ofuseful drug candidates is usually the analysis of the relationshipbetween the chemical structure of a hit compound and its biological orpharmacological activity. Molecular structure and biological activityare correlated by observing the results of systemic structuralmodification on defined biological endpoints. Structure-activityrelationship information available from the first round of screening canthen be used to generate small secondary libraries which aresubsequently screened for compounds with higher affinity. The process ofperforming synthetic modifications of a biologically active compound tofulfill stereoelectronic, physicochemical, pharmacokinetic, andtoxicologic factors required for clinical usefulness is called leadoptimization.

The candidate compounds identified by the screening methods of theinvention can similarly be subjected to a structure-activityrelationship analysis, and chemically modified to provide improved drugcandidates. The present invention also encompasses these improved drugcandidates.

C—Identification of Regulators of Iron Metabolism and Regulators ofHepcidin Expression

According to the screening methods of the present invention,determination of the ability of a candidate compound to regulate ironmetabolism or to regulate hepcidin expression includes comparison of atleast one factor that is representative of the signaling activity of aTGF-β superfamily member measured in the test and control systems.

In the inventive screening methods, a candidate compound is identifiedas a regulator of hepcidin expression or activity and/or as a regulatorof iron metabolism, if the factor measured in the test system is less orgreater than the factor measured in the control system. Morespecifically, if a candidate compound is found to be an agonist of TGF-βor an antagonist of BMP, it is identified as an inhibitor of hepcidinexpression or activity and/or as an enhancer of iron metabolism.Alternatively, if a candidate compound is found to be an antagonist ofTGF-β or an agonist of BMP, it is identified as an enhancer of hepcidinexpression or activity and/or as an inhibitor of iron metabolism.

Factors representative of the signaling activity of a TGF-β superfamilymember include reporter assay signaling, and target gene expression(e.g., extracellular matrix protein genes). Other factors representativeof the signaling activity of a TGF-β superfamily member include Smadphosphorylation, translocation of phosphorylated Smad proteins to thenucleus, and alterations in cell growth rates. In certain embodiments,the factor measured in the screening methods of the invention is theamount of iron present in the system. In other embodiments, the factormeasured is the level of hepcidin mRNA expression in the system. Instill other embodiments, the factor measured is the hepcidin/actin ratioin the system.

Reproducibility of the results obtained in the inventive screeningmethods may be tested by performing the analysis more than once with thesame concentration of the same candidate compound (for example, byincubating cells in more than one well of an assay plate). Additionally,since candidate compounds may be effective at varying concentrationsdepending on the nature of the compound and the nature of itsmechanism(s) of action, varying concentrations of the candidate compoundmay be tested (for example, different concentrations can be added todifferent wells containing cells). Generally, candidate compoundconcentrations from 1 fM to about 10 mM are used for screening.Preferred screening concentrations are generally between about 10 pM andabout 100 μM.

In certain embodiments, the methods of the invention further involve theuse of one or more negative and/or positive control compounds. Apositive control compound may be any molecule or agent that is known tomodulate the signaling activity of the TGF-β family member studied inthe screening assay. A negative control compound may be any molecule oragent that is known to have no effect on the signaling activity of theTGF-β family member studied in the screening assay. In theseembodiments, the inventive methods further comprise comparing themodulating effects of the candidate compound to the modulating effects(or absence thereof) of the positive or negative control compound. Forexample, Noggin and decorin may be used as positive controls for theinhibition of BMP signaling and TGF-β signaling, respectively.

D—Characterization of Candidate Compounds

As will be appreciated by those skilled in the art, it is generallydesirable to further characterize regulators identified by the inventivescreening methods.

For example, if a candidate compound has been identified as a modulatorof the signaling activity in a given TGF-β superfamily member in a givencell culture system (e.g., an established cell line), it may bedesirable to test this ability in a different cell culture system (e.g.,primary or secondary cells). Alternatively or additionally, it may bedesirable to directly evaluate the effects of the candidate compound onhepcidin expression, for example by quantitating hepcidin mRNAexpression using real-time quantitative RT-PCR (as described in Example1). It may also be desirable to evaluate the specificity of thecandidate compound by testing its ability to modulate the signalingactivity of other members of the TGF-β superfamily members. It may alsobe desirable to perform pharmacokinetics and toxicology studies.

Candidate compounds identified by screening methods of the invention mayalso be further tested in assays that allow for the determination of thecompounds' properties in vivo. Suitable animal models include animalmodels that can exhibit iron deficient or iron overload or that havebeen determined to exhibit up-regulation of hepcidin expression ordown-regulation of hepcidin expression.

Examples of animal models for iron overload include, but are not limitedto, mice treated with carbonyl iron, β2-microglobulin knockout mice (C.Pigeon et al., J. Biol. Chem., 2001, 276: 7811-7819), USF2 (UpstreamStimulatory Factor 2) knockout mice (G. Nicolas et al., Proc. Natl.Acad. Sci. USA, 2001, 98: 8780-8785), and HFE knockout mice (K. A. Ahmadet al., Blood Cells Mol. Dis., 2002, 29: 361-366). Examples of animalmodels for iron deficiency include, but are not limited to, models ofanemia in mice with acute hemolysis, provoked by phenylhydrazine, andmice with bleeding provoked by repeated phlebotomies (G. Nicolas et al.,J. Clin. Invest., 2002, 110: 1037-1044). Examples of animal modelsexhibiting increased hepcidin mRNA expression include mice treated bypartial hepatectomy (N. Kelley-Loughnane et al., Hepatology, 2002, 35:525-534), by lipopolysaccharide (G. R. Lee, Semin. Hematol., 1983, 20:61-80), and turpentine (G. Nicolas et al., J. Clin. Invest., 2002, 110:1037-1044).

E—Pharmaceutical Compositions of Identified Regulators

The present invention also provides pharmaceutical compositions, whichcomprise, as active ingredient, an effective amount of at least oneregulator of iron metabolism or at least one regulator of hepcidinexpression or activity identified by an inventive screening assay. Thepharmaceutical compositions of the invention may be formulated usingconventional methods well known in the art. Such compositions include,in addition to the active ingredient(s), at least one pharmaceuticallyacceptable liquid, semiliquid or solid diluent acting as pharmaceuticalvehicle, excipient or medium, and termed here “pharmaceuticallyacceptable carrier”.

According to the present invention, pharmaceutical compositions mayinclude one or more regulators of the invention as active ingredients.Alternatively, a pharmaceutical composition containing an effectiveamount of one inventive regulator may be administered to a patient incombination with or sequentially with a pharmaceutical compositioncontaining a different inventive regulator. However, in both cases, theregulators preferably have the same regulatory effect on iron metabolismand/or hepcidin expression or activity. For example, an agonist of BMPand an antagonist of TGF-β, which both enhance hepcidin expression andinhibit iron metabolism, may be administered to a subject in a singlepharmaceutical composition, or in two different pharmaceuticalcompositions.

As will be appreciated by one skilled in the art, a regulator ofhepcidin expression or an iron metabolism regulator, or a pharmaceuticalcomposition thereof, may be administered serially or in combination withconventional therapeutics used in the treatment of iron metabolismdisorders. Such therapeutics include iron supplements (in the case ofdiseases associated with iron deficiency) and iron chelating agents (inthe case of diseases associated with iron overload). Iron supplementsinclude, but are not limited to, ferrous fumarate, ferrous gluconate,ferrous sulfate, iron dextran, iron polysaccharide, iron sorbitol,sodium ferric gluconate, and iron sucrose. Iron chelating agentsinclude, for example, desferrioxamine, bathophenanthroline, andClioquinol. Iron supplements or iron chelating agents may be included inpharmaceutical compositions of the present invention. Alternatively,they may be administered in separate pharmaceutical compositions.

Alternatively or additionally, a regulator of hepcidin expression or aniron metabolism regulator, or a pharmaceutical composition thereof, maybe administered serially or in combination with conventional therapeuticregimens for the treatment of iron metabolism disorders. These include,for example, phlebotomy, in the case of conditions associated with ironoverload.

III. Methods of Treatment

In another aspect, the present invention provides methods for thetreatment and/or prevention of conditions associated with perturbationsin iron metabolism, including conditions associated with iron overloadand conditions associated with iron deficiency. These methods compriseadministering to a subject having or at risk or having such a condition,an effective amount of a compound that modulates the signaling activityof at least one TGF-β superfamily member, wherein modulation of thesignaling activity of the TGF-β superfamily member results in regulationof hepcidin expression or activity in the subject.

The compound may be a known agonist or antagonist of the TGF-βsuperfamily member. Alternatively, the compound may be a regulator ofiron metabolism or a regulator of hepcidin expression identified, forexample, by a screening method provided by the present invention.

A—Iron Metabolism Diseases

Conditions that may be treated and/or prevented using the methods of thepresent invention include any disease, disorder, or syndrome associatedwith perturbations in iron metabolism. Perturbations in iron metabolismmay be associated with disturbances in one or more of iron uptake, ironabsorption, iron transport, iron storage, iron processing, ironmobilization, and iron utilization. Generally, perturbations in ironmetabolism result in iron overload or iron deficiency.

Conditions associated with iron overload include both primary andsecondary iron overload diseases, syndromes or disorders, including, butnot limited to, hereditary hemochromatosis, porphyria cutanea tarda,hereditary spherocytosis, hyprochromic anemia, hysererythropoieticanemia (CDAI), faciogenital dysplasia (FGDY), Aarskog syndrome,atransferrinemia, sideroblastic anemia (SA), pyridoxine-responsivesidero-blastic anemia, and hemoglobinopathies such as thalassemia andsickle cell. Some studies have suggested an association between ironmetabolism disorders, such as thalassemia and hemochromatosis, and anumber of disease states, such as type II (non-insulin dependent)diabetes mellitus and atherosclerosis (A. J. Matthews et al., J. Surg.Res., 1997, 73: 35-40; T. P. Tuomainen et al., Diabetes Care, 1997, 20:426-428).

Diseases associated with iron deficiency include, but are not limitedto, anemia of chronic disease, iron deficiency anemias, functional irondeficiency, and microcytic anemia. The term “anemia of chronic disease”refers to any anemia that develops as a result of, for example, extendedinfection, inflammation, neoplastic disorders, etc. The anemia whichdevelops is often characterized by a shortened red blood cell life spanand sequestration of iron in macrophages, which results in a decrease inthe amount of iron available to make new red blood cells. Conditionsassociated with anemia of chronic disease include, but are not limitedto, chronic bacterial endocarditis, osteomyelitis, rheumatic fever,ulcerative colitis, and neoplastic disorders. Further conditions includeother diseases and disorders associated with infection, inflammation,and neoplasms, including, for example, inflammatory infections (e.g.,pulmonary abscess, tuberculosis, etc), inflammatory noninfectiousdisorders (e.g., rheumatoid arthritis, systemic lupus erythrematosus,Crohn's disease, hepatitis, inflammatory bowel disease, etc.), andvarious cancers, tumors, and malignancies (e.g., carcinoma, sarcoma,lymphoma, etc.). Iron deficiency anemia may result from conditions suchas pregnancy, menstruation, infancy and childhood, blood loss due toinjury, etc.

It has also been suggested that iron metabolism plays a role in a numberof other diseases states, including cardiovascular disease, Alzheimer'sdisease, Parkinson's disease, and certain types of colo-rectal cancers(see, for example, P. Tuomainen et al., Circulation, 1997, 97:1461-1466; J. M. McCord, Circulation, 1991, 83: 1112-1114; J. L.Sullivan, J. Clin. Epidemiol., 1996, 49: 1345-1352; M. A. Smith et al.,Proc. Nat. Acad. Sci., 1997, 94: 9866-9868; P. Riederer et al., J.Neurochem., 1989, 512: 515-520; P. Knekt et al., Int. J. Cancer, 1994,56: 379-382).

B—Subject Selection

Subjects suitable to receive a treatment according to the inventivemethods include individuals that have been diagnosed with a conditionassociated with perturbations in iron metabolism, including, but notlimited to, the diseases and disorders listed above, and individualsthat are susceptible to conditions associated with perturbations in ironmetabolism. Suitable subjects may or may not have previously receivedtraditional treatment for the condition.

Other suitable subjects are individuals that exhibit iron deficiency oriron overload. Iron overload and iron deficiency may be detected using anumber of laboratory tests available in the art that allow for thedetermination of total iron-binding capacity (TIBC), levels of serumiron, ferritin, hemoglobin, hematocrit, and urinary creatinine.

C—Administration

A treatment according to methods of the present invention may consist ofa single dose or a plurality of doses over a period of time. A regulatorof hepcidin expression or modulator of iron metabolism, or apharmaceutical composition thereof, may also be released from a depotform per treatment. The administration may be carried out in anyconvenient manner such as by injection (subcutaneous, intravenous,intramuscular, intraperitoneal, or the like), oral administration, orsublingual administration.

Effective dosages and administration regimens can be readily determinedby good medical practice and the clinical condition of the individualpatient. The frequency of administration will depend on thepharmacokinetic parameters of the compound and the route ofadministration. The optimal pharmaceutical formulation can be determineddepending upon the route of administration and desired dosage. Suchformulations may influence the physical state, stability, rate of invivo release, and rate of in vivo clearance of the administeredcompounds.

Depending on the route of administration, a suitable dose may becalculated according to body weight, body surface area, or organ size.Optimization of the appropriate dosage can readily be made by thoseskilled in the art in light of pharmacokinetic data observed in humanclinical trials. The final dosage regimen will be determined by theattending physician, considering various factors which modify the actionof drugs, e.g., the drug's specific activity, the severity of the damageand the responsiveness of the patient, the age, condition, body weight,sex and diet of the patient, the severity of any present infection, timeof administration and other clinical factors. As studies are conducted,further information will emerge regarding the appropriate dosage levelsand duration of treatment for various conditions associated with ironoverload and iron deficiency.

EXAMPLES

The following examples describe some of the preferred modes of makingand practicing the present invention. However, it should be understoodthat these examples are for illustrative purposes only and are not meantto limit the scope of the invention. Furthermore, unless the descriptionin an Example is presented in the past tense, the text, like the rest ofthe specification, is not intended to suggest that experiments wereactually performed or data were actually obtained.

Some of the results presented in this section have been described by theApplicants in a recent scientific manuscript (J. L. Babitt et al., “BoneMorphologenetic Protein Signaling by Hemoguvelin Regulates HepcidinExpression”, submitted to Nature Genetics on Feb. 3, 2006). Thismanuscript is incorporated herein by reference in its entirety.

Example 1 Effects of BMP and TGF-β on Hepcidin Transcription in LiverCells

Study Protocol. The effects of BMP-2, Noggin (a well-known BMPinhibitor), and TGF-β1 on hepcidin mRNA expression in HepG2 liverhepatoma cells were studied and quantitated using real-time quantitativeRT-PCR.

HepG2 cells (ATTC Number HB-8065) were grown in a-MEM (Minimal EssentialMedium Alpha Medium with L-Glutamine supplemented with 10% fetal bovineserum, 100 U/mL penicillin and 100 μg/mL streptomycin) to 60% confluenceon 6 cm tissue culture plates. Cells were then incubated in low-serumconditions (a-MEM with 1% FBS), with 1 μg/mL Noggin.Fc (R & D Systems,Minneapolis, Minn.) at 37° C. for 48 hours, or serum-starved for 6 hoursfollowed by incubation with 50 ng/mL BMP-2 (R & D Systems) at 37° C. for16 hours, or with 1 ng/mL TGF-β1 (R & D Systems) at 37° C. for 16 hours.Alternatively, cells were incubated at 37° C. for 72 hours withnon-transferrin-bound iron (65 μM Fe-NTA). Fe-NTA was generated bycombining 1:1 molar ratio of FeCI₃ hexahydrate (Sigma) in 0.1 molar HClwith nitrilotriacetic acid (NTA, Sigma) in a-MEM medium supplementedwith 20 mM Hepes pH 7.5, as previously described (E. W. Randell et al.,J. Biol. Chem., 1994, 269: 16046-16053; S. G. Gehrke et al., Blood,2003, 102: 371-376).

Total RNA was isolated from HepG2 cells treated as described above usingthe RNAeasy Mini Kit (QIAGEN™, Valencia, Calif.) including DNAsedigestion with the RNAse-free DNAse Set (QIAGEN™), according to themanufacturer's instructions. Real-time quantification of mRNAtranscripts was performed using a 2-step reverse transcriptasepolymerase chain reaction (RT-PCR) using the ABI PRISM® 7900HT SequenceDetection System and SDS software version 2.0. First strand cDNAsynthesis was performed using ISCRIPT™ cDNA Synthesis Kit (BIORAD™Laboratories, Hercules, Calif.) according to the manufacturer'sinstructions using 2 μg of total RNA template per sample. In a secondstep, transcripts of hepcidin were amplified with sense primer HepcF(5′-CTGCAACCCCAGGACAGAG-3′ (SEQ ID NO: 1)) and antisense primer HepcR(5′-GGAATAAATAAGGAAGGGAGGGG-3′ (SEQ ID NO: 2)) and detected using ITAQ™SYBR Green Supermix with ROX (BIORAD™). In parallel, transcripts ofβ-actin were amplified with sense primer BactF(5′-AGGATGCAGAAGGAGATCACTG-3′ (SEQ ID NO: 3)) and antisense primer BactR(5′-GGGTGTAACGCAACTAAGTCATAG-3′ (SEQ ID NO: 4)) and detected in asimilar mariner to serve as an internal control.

Standard curves for hepcidin and 13-actin were generated from accuratelydetermined dilutions of cDNA clones of hepcidin (IMAGE clones 4715540)and β-actin (IMAGE clone 3451917) as templates (IMAGE clones werepurchased from Open Biosystems, and DNA sequenced to verify theirinserts). Samples were analyzed in triplicate, and results are reportedas the ratio of mean values for hepcidin to β-actin:

Results. As shown in FIG. 1, incubation of HepG2 cells with Fe-NTAdecreased the hepcidin/actin ratio expression approximately 6-fold (bar4), a result which correlates well with a previously reported decreasein hepcidin expression caused by Fe-NTA in HepG2 cells (S. G. Gehrke etal., Blood, 2003, 102: 371-376). Significantly, incubation with 50 ng/mLBMP-2 increased the hepcidin/actin ratio expression by 10-fold (±8%)over baseline (compare bar 2 with bar 1). In contrast, incubation with 1μg/mL Noggin.Fc (which inhibits endogenous BMP signaling) decreasedhepcidin/actin ratio expression 50-fold (±19%) below baseline (comparebar 3 with bar 1).

As shown in FIG. 2, incubation of HepG2 cells with 50 ng/mL BMP-2increased the hepcidin/actin ratio expression by 15-fold (±8%) overbaseline (compare bar 2 with bar 1). In contrast, incubation with 1μg/mL Noggin.Fc (which inhibits endogenous BMP signaling) decreasedhepcidin/actin ratio expression 6-fold (±23%) below baseline (comparebar 3 with bar 1). In addition, incubation with 1 ng/ml TGF-β1 decreasedhepcidin/actin ratio expression 6-fold (±6%) below baseline (compare bar4 with bar 1).

Example 2 HJV.Fc Protein as Modulator of Hepcidin Expression

Juvenile hemochromatosis is a severe variant of hemochromatosis causedby mutations in two genes that give indistinguishable phenotypes. Onegene encodes hepcidin (RAMP, 19q13.1). The second gene has recently beenidentified as hemojuvelin (HJV, 1q21). Although the function of HJV isunknown, hepcidin levels are depressed in persons with HJV mutations,indicating that HJV may be a modulator of hepcidin expression. Asalready mentioned herein, HJV is also a member of the repulsive guidancemolecule (RGM) family of proteins, including RGMa and DRAGON, neuronaladhesion molecules which were recently shown by the present Applicantsto function as a BMP co-receptor (J. L. Babitt et al., J. Biol. Chem.,2005, 280: 29820-29827; T. A. Samad et al., J. Biol, Chem., 2005, 280:14122-14129, each of which is incorporated herein by reference in itsentirety). The study presented below was undertaken to investigatewhether HJV could similarly mediate BMP signaling.

Materials and Methods

cDNA subcloning. cDNA encoding mutant murine HJV with a glycine tovaline substitution at amino acid 313 (mHJVG313V) was generated by anoverlapping PCR strategy. Two primers

-   5′-ACCGAATTCGGGGGACCTGGCTGGATAG-3′ (SEQ ID NO: 5) and-   5′-CGGAGGGCATACCCCAACACACAG-3′ (SEQ ID NO: 6) were used to generate    an N-terminal fragment of mHJV incorporating a substitution of    valine for glycine at amino acid 313. Primers    5′-CTGTGTGTTGGGGtATGCCCTCCG-3′ (SEQ ID NO: 7) and-   5′-CCCTCTAGATGGTGCCAGTCTCCAAAAGC-3′ (SEQ ID NO: 8) were used to    generate a C-terminal fragment of HJV with the identical    substitution. A final round of PCR was performed using the outside    primers to generate mutant mHJVG313V, which was then subcloned into    the expression vector pCDNA 3.1 (INVITROGEN™, Carlsbad, Calif.).

cDNA encoding soluble mHJV.Fc fusion protein was generated by PCR of theextracellular domains of wild-type murine HJV using primers:5′-GGAAGCTTATGGGCCAGTCCCCTAGT-3′ (SEQ ID NO: 9) and

-   5′-CCGGATCCGCTAAGTTCTCTAAATCCGTC-3′ (SEQ ID NO: 10), followed by    subcloning into the mammalian expression vector plgplus (R & D    Systems, Minneapolis, Minn.) in-frame with the Fc portion of human    IgG.

cDNA encoding flag-tagged human HJV (hHJV) was generated from human HJVtranscript variant B (IMAGE clone 6198223), which does not contain exon2, purchased form ATCC (#10642497). Exon 2 which codes for the signalpeptide of the full length HJV isoform (variant A, ACCESSION #NM213653)was amplified by PCR from human genomic DNA using the forward primer:5′-CGAGAATTCACTTACAGGGCTIVCGGTCA-3′ (SEQ ID NO: 11) and the reverseprimer: 5′-GCATTGAGAATGAGCATGTCCACAGAGGAGCAGCAG-3′ (SEQ ID NO: 12). Adownstream cDNA fragment corresponding to the rest of the codingsequence (including the stop codon) was amplified by PCR from the IMAGEclone using the forward primer5′-CCTCTGTGGACATGCTCATTCTCAATGCAAGATCCTCCGCTG-3′ (SEQ ID NO: 13) and5′-CGTCTCGAGTTACTGAATGCAAAGCCACAGAACAAAGAGC-3′ (SEQ ID NO: 14), asreverse primer. The two overlapping fragments were fused together by PCRand the full length cDNA product was then cut with EcoR I and Xho I andclone into pCDNA3.1 (INVITROGEN™), to generate pCDNA3.1-hHJV. Togenerate N-terminal Flag-tagged hHJV, an upstream fragment correspondingto the beginning of exon 3 was generated by PCR using forward primer:

-   5′-GACAGATCTGCGGCCGCTCATTCTCAATGCAAGATCCTCCG-3′ (SEQ ID NO: 15), and    reverse primer: 5′-GAGCAGTTGTGCTGGATCATCAGG-3′ (SEQ ID NO: 16).    Following a Not I /Sac II digestion, the fragment was ligated    together with a downstream Sac II/Xba I hHJV fragment, removed from    pCDNA3.1-hHJV, into Not I/XbaI sites of p3XFLAGCMV9    (SIGMA-ALDRITCH®).

cDNA encoding mutant Flag-tagged hHJV G99V (hG99V), with a valine toglycine substitution at amino acid 99, was generated from hHJV by sitedirected mutagenesis using the QUIKCHANGE® kit (STRATAGENE™, La Jolla,Calif.). cDNA encoding the hepcidin promoter luciferase construct wasgenerated by subcloning the −2649 to +45 region of the human hepcidinpromoter 46 in the pGL2-Bsic vector (PROMEGA™, Madison, Wis.) upstreamof the firefly luciferase reporter gene. All cDNA's were sequenced toverify the fidelity of the constructs (MGH, Molecular Biology DNASequencing Core Facility).

Cell culture and transfection. CHO cells (American Type CultureCollection ATCC #CCL-61) were cultured in F-12K Nutrient Mixture,Kaighn's Modification (INVITROGEN™) supplemented with 10% fetal bovineserum (FBS) (Atlanta Biologicals, Lawrenceville, Ga.). HepG2 cells andHep3B cells (ATCC #HB-8065 and #HB-8064) were cultured in MinimalEssential Alpha Medium with L-glutamine (a-MEM, INVITROGEN™) containing10% FBS. HEK 293 cells (ATCC #CRL-1573) were cultured in Dulbecco'smodification of Eagle's medium (DMEM; CELLGRO® Mediatech, Herndon, Va.)supplemented with 10% FBS. All plasmid transfections were performed withLipofectamine 2000 (INVITROGEN™) or Effectene transfection reagent(QIAGEN™ Inc, Valencia, Calif.) according to manufacturer instructions.Stably transfected cells were selected and cultured in 1 mg/ml Geneticin(CELLGRO® Mediatech, Herndon, Va.).

Luciferase assay. HepG2 or Hep3B cells were transiently transfected with2.5 μg BMP responsive luciferase reporter (BRE-Luc), 2.5 μg TGF-βresponsive luciferase reporter, (CAGA)₁₂MPL-Luc (CAGA-Luc) (both kindlyprovided by Peter ten Dijke, Leiden University Medical Center, TheNetherlands), or 2.5 μg hepcidin promoter luciferase reporter construct,in combination with 0.25 μg pRL-TK Renilla luciferase vector (Promega)to control for transfection efficiency, with or without co-transfectionwith wild-type or mutant HJV cDNA. Forty-eight hours after transfection,cells were serum starved in a-MEM supplemented with 1% FBS for 6 hoursand treated with varying amounts of TGF-β1 or BMP ligands (R & DSystems) for 16 hours, in the absence or presence of 1 μg/ml noggin (R &D Systems) or 20 μg/ml neutralizing anti-BMP-2/4 antibody (R & DSystems). Cells were lysed, and luciferase activity was determined withthe Dual Reporter Assay according to the manufacturer's instructions(PROMEGA™). Experiments were performed in duplicate or triplicate wells.Relative luciferase activity was calculated as the ratio of firefly(reporter) and Renilla (transfection control) luciferase values, and isexpressed as the fold increase over unstimulated cells transfected withreporter alone.

Purification of mHJV.Fc. CHO cells stably expressing mHJV.Fc werecultured in F-12K Nutrient Mixture, Kaighn's Modicfication, supplementedwith 5% ultra-low IgG FBS (Invitrogen) using 175-cm² multifloor flasks(Denville Scientific, Southplainfield, N.J.). mHJV.Fc was purified fromthe media of stably transfected cells via one-step Protein A affinitychromatography using HiTrap rProtein A FF columns (Amersham Biosciences,Piscataway, N.J.) as previously described (E. del Re et al., J. Biol.Chem., 2004, 279: 22765-22772, which is incorporated herein by referencein its entirety). Purified protein was eluted with 100 mM glycine-HCI,pH 3.2 and neutralized with 0.3 M Tris-HCI pH 9 as previously described(E. del Re et al., J. Biol. Chem., 2004, 279: 22765-22772). mHJV.Fc wassubjected to reducing sodium dodecylsulfate-polyacrylamide gelelectrophoresis (SDS-PAGE) and gels were stained with Bio-safe Coomassieblue (Bio-Rad, Hercules, Calif.) to determine purity and quantifyprotein concentration.

Generation ofHIV antibody and immunoblot analysis. An affinity purifiedrabbit polyclonal anti-murine HJV antibody (αHJV) was raised against thepeptide RVAEDVARAFSAEQDLQLC (SEQ ID NO: 17), amino acids 292-310 in theC-terminus of murine HJV upstream of its hybrophobic tail (G.Papamokolaou et al, Nat. Genet., 2004, 36: 77-82). Livers from129S6/SvEvTac wild-type or Hjv−/− mice (F. W. Huang et al., J. Clin,Invest., 2005, 115: 2187-2191), or cells transfected with wild-type ormutant HJV, were homogenized/sonicated in lysis buffer (200 mM Tris-HCI,pH 8, 100 mM NaCl, 1 mM EDTA, 0.5% NP-40, and 10% glycerol) containing amixture of protease inhibitors (Roche, Mannheim, Germany) as previouslydescribed (J. L. Babitt et al., J. Biol. Chem., 2005, 280: 29820-29827,which is incorporated herein by reference in its entirety). For assaysexamining phosphorylated Smad expression, 1 mM sodium orthovanadate(Sigma, St. Louis, Mo.) and 1 mM sodium fluoride (SIGMA-ALDRITCH®) wereadded to the lysis buffer as phosphatase inhibitors. Purified mHJV.Fc,transfected cell lysates, or liver lysates, were subjected to reducingsodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) andWestern blot as previously described (J. L. Babitt et al., J. Biol.Chem., 2005, 280: 29820-29827) using HJV antibody (1:1000, 4 mg/mL) at4° C. overnight, goat anti-human Fc antibody (1:1000) (JacksonImmunoResearch Laboratories, West Grove, Pa.) at room temperature for 1hour, or rabbit polyclonal anti-phosphosmad1/5/8 antibody (1:1000) (CellSignaling, Beverly, Mass.) at 4° C. overnight. Blots were stripped andre-probed with mouse monoclonal anti-β-actin antibody (1:5000) (clone AC15, SIGMA-ALDRITCH®), rabbit polyclonal anti-Smad1 antibody (1:250)(Upstate Biotechnology, Lake Placid, N.Y.) at 4° C. overnight, or rabbitpolyclonal anti-actin antibody (1:50) (Biomedical Technologies, Inc.,Stoughton, Mass.) for room temperature at 1 hour as loading controls.

HJV antibody recognizes major bands at ˜49 kDa and ˜30 kDA in the liver(lane 2, FIG. 3(A)), corresponding to the predicted size of full-lengthHJV and HJV which has been cleaved at a previously described proteolyticcleavage site. The ˜62 kDa band likely represents a higher order form.No bands were seen after pre-incubation of antibody with competingpeptide (lane 1). Similar results were seen in transfected CHO cellswith the difference in size likely due to differential glycosylation oraltered processing (FIG. 3(B)). HJV.Fc cDNA, generated by fusing theextracellular domain of HJV with human Fc, was stably transfected intoCHO cells, and HJV.Fc protein was purified from the media by one-stepprotein A chromatography. Western Blot of purified protein with anti-HJVantibody (lanes 1-2) or anti-Fc antibody (lanes 34) confirmed thepresence of both domains in the purified protein and provided furthervalidation of the anti-HJV antibody. Both antibodies recognized ˜70-75kDa and ˜60 kDa bands, corresponding to the predicted sizes of thefull-length and proteolytically cleaved proteins. A lower band at ˜40-45kDa suggests another possible proteolytic cleavage site.

Ligand iodination and crosslinking. Two (2) μg of carrier-free humanBMP-2 or BMP-4 ligand (R & D Systems) per reaction was iodinated with[¹²⁵I] by the modified chloramine-T method as previously described (C.A. Frolick et al., J. Biol. Chem., 1984, 259: 10995-11000, which isincorporated herein by reference in its entirety). ¹²⁵I-BMP-2 wasincubated with 60 ng mHJV.Fc or ALK5.Fc (R & D Systems) in 20 mM HEPES(pH 7.8) with 0.1% BSA and a mixture of protease inhibitors (RocheDiagnostics) or with buffer alone. This mixture was incubated in theabsence or presence of 2.5 M disuccinimidyl suberate (DSS, Sigma, St.Louis, Mo.) followed by incubated with Protein A Sepharose beads(Amersham) as previously described (J. L. Babitt et al., J. Biol. Chem.,2005, 280: 29820-29827). Beads were washed with phosphate bufferedsaline (PBS) and protein eluted by non-reducing Laemmli sample buffer(Bio-Rad). Eluted protein was separated by SDS-PAGE and analyzed byautoradiography.

Quantitative reverse transcription polymerase chain reaction (RT-PCR).HepG2 or Hep3B cells were grown to 60% confluence on 6 cm tissue cultureplates. Where indicated, cells were transfected with varying amounts ofhHJV or hG99V cDNA. Twenty-four (24) hours after transfection, cellswere serum-starved in a-MEM with 1% FBS followed by incubation with 50ng/mL BMP-2 at 37° C. for various times or with 1 μg/mL noggin at 37° C.for 48 hours. For cycloheximide experiments, 10 μg/ml cycloheximide wasadded for 30 minutes prior to addition of BMP-2. Total RNA was isolatedusing the RNeasy Mini Kit (QIAGEN™ Inc., Valencia Calif.), includingDNAse digestion with the RNase-Free DNase Set (QIAGEN)™ according to themanufacturer's instructions. Real time quantification of mRNAtranscripts was performed using a 2-step reverse transcriptasepolymerase chain reaction (RT-PCR) using the ABI PRISM® 7900HT SequenceDetection System and SDS software version 2.0. First strand cDNAsynthesis was performed using ISCREPT™ cDNA Synthesis Kit (BIORAD™)according to the manufacturer's instructions using 2 μg total RNAtemplate per sample.

In a second step, transcripts of hepcidin were amplified with senseprimer HepcF 5′-CTGCAACCCCAGGACAGAG-3′ (SEQ ID NO: 1) and antisenseprimer HepcR 5′-GGAATAAATAAGGAAGGGAGGGG-3′ (SEQ ID NO: 2) and detectedusing ITAQ™ SYBR Green Superrnix with ROX (BIORAD™) according to themanufacturer's instructions. In parallel, transcripts of β-actin wereamplified with sense primer BactF 5′-AGGATGCAGAAGGAGATCACTG-3′ (SEQ IDNO: 3) and antisense primer 5′-GGGTGTAACGCAACTAAGTCATAG-3′ (SEQ ID NO:4) and detected in a similar manner to serve as an internal control.Standard curves for hepcidin and β-actin were generated from accuratelydetermined dilutions of plasmids containing cDNA sequences of hepcidinand β-actin as templates (IMAGE clones 4715540 and 3451917 from OpenBiosystems followed by sequence analyses to verify the proposed insert).Samples were analyzed in triplicate, and results are reported as theratio of mean values for hepcidin to β-actin. Transcripts for BMP-2 andBMP-4 were amplified from HepG2 cDNA generated above using the forwardprimer 5′-CGTGACCAGACITTTGGACAC-3′ (SEQ ID NO: 18) and reverse primer5′-GGCATGATTAGTGGAGTIVAG-3′ (SEQ ID NO: 19) (for BMP-2) and the forwardprimer: 5′-AGCAGCCAAACTATGGGCTA-3′ (SEQ ID NO: 20) and reverse primer5′-TGGTTGAGTTGAGGTGGICA-3′ (SEQ ID NO: 21) (for BMP-4).

Primary Hepatocyte Isolation and Culture. Primary hepatocytes wereisolated by collagenase digestion of livers from 8 to 10 week old129S6/SvEvTac wild-type or Hjv−/− mice (F. W. Huang et al., J. Clin.Invest., 2005, 115: 2187-2191) using previously described methods (J.Lin et al., Cell, 2004, 119: 121-135, which is incorporated herein byreference in its entirety). Briefly, mice were perfused through theinferior vena cava with calcium-free Hank's Balanced Salt Solution(HBSS) (Mediatech Inc.) supplemented with 0.5 mM EDTA and 16.7 mM sodiumbicarbonate for 4 minutes at a rate of ˜1.5 mL/min. Mice weresubsequently perfused with calcium-containing HBSS containing 0.05%collagenase (SIGMA-ALDRITCH®), 1% bovine serum albumin and 16.7 mMsodium bicarbonate for 8 min. After enzymatic digestion, hepatocyteswere liberated into culture medium [1:1 Dulbecco's modifiedEagle's/Ham's F12 medium (GIBCO™, Grand Island, N.Y.) supplemented with100 IU/ml penicillin, 100 μg/ml streptomycin, 18 mM HEPES, 1 mM sodiumpyruvate, 10 μg/ml insulin, 5.5 μg/ml transferrin, and 5 ng/ml selenium(ITS; SIGMA-ALDRITCH®), 2 mM L-glutamine, 0.1 mM non-essential aminoacids (Gibco™), 10% FBS (HYCLONE™, Logan Utah)], passed through a 100 μmBD FALCON™ mesh cell strainer (BD Biosciences, San Jose Calif.),centrifuged, gently washed with culture medium, and counted.

Cells (>90% hepatocytes by microscopy) were seeded on collagen-coatedplates (SIGMA-ALDRITCH®) at 5×10⁵ cells/60 mm dish. After 2 to 3 hours,cells were washed with PBS, serum starved with culture medium containing1% FBS for 6 hours, and stimulated with recombinant human BMP-2 atvarying concentrations for 12 hours. RNA was isolated using the RNeasykit according to manufacturer's directions (QIAGEN™).

Northern Blot Analysis. Total RNA (2.5 μg) from primary hepatocytes wasseparated on a 1% formaldehyde agarose gel and transferred onto HybondN+ membranes (Amersham Pharmacia Biotech). Membranes were baked for twohours at 80° C. under vacuum and hybridized with radioactively labeledprobes specific for mouse hepcidin 1 amplified from Soares mousep3NMF19.5 Mus musculus cDNA IMAGE clone: 317863 with primers5′-TCCTTAGACTGCACAGCAGAA-3′ (SEQ ID NO: 22) and5′-ATAAATAAGGACGGGAGGGG-3′ (SEQ ID NO: 23) and β-actin (S. Alonso etal., I. MoI. Evol., 1986, 23: 11-22). Expression was quantified using aphosphorimager (Molecular Dynamics, now Amersham Biosciences) andnormalized to β-actin or 28S RNA as loading controls.

Statistical Analysis. A two-tailed Student's t-test was used with a Pvalue of <0.05 to determine statistical significance.

Result 1: HJY Induces BMP but not TGF-β Signals

HepG2 cells were transfected with a BMP-responsive luciferase reporter(BRE-Luc, FIG. 4, panels A and C) or TGF-β responsive luciferasereporter (CAGA-Luc, FIG. 4, panel B) either alone or in combination withcDNA encoding HJV. Transfected cells were then incubated with or without0.5 nM BMP-2, BMP-4, or 40 pM TGF-β1 for 16 hours followed bymeasurement of luciferase activity. Stimulation with BMP or TGF-β3increased the relative luciferase activity for their respectivereporters compared with unstimulated cells (A and B, compare bars 2 to1). Co-transfection with HJV similarly increased BRE luciferase activityeven in the absence of exogenous BMP stimulation (A, bar 3).HJV-mediated BMP signaling was dose dependent (C, grey bars), and thepresence of HJV augmented signaling produced by exogenous BMP (C, blackbars). In contrast, co-transfection with HJV (up to 1 μg) did notincrease CAGA-luciferase activity above baseline (B, bar 3). Takentogether, these results demonstrate that HJV can mediate BMP signalingbut not TGF-β signaling, and that HJV behaves in a manner consistentwith a possible accessory receptor for BMP-2.

Result 2: HJV Mediated BMP Signaling is Inhibited by Noggin

The ability of HJV to mediate BMP signaling even in the absence ofexogenous BMP ligand raises the question of whether HJV is acting in aligand-independent manner, or whether it is augmenting signaling byendogenous BMP ligands. Studies were therefore undertaken to determinewhether HJV-mediated signaling could be inhibited by Noggin, a solubleinhibitor of BMP signaling that functions by binding to BMP ligands andblocking the binding epitopes for BMP receptors.

HepG2 cells were co-transfected with BRE-Luc and HJV cDNA or emptyvector. Transfected cells were incubated with or without 0.5 nMexogenous BMP-2 in the presence or absence of 1 μg Noggin protein for 16hours followed by measurement of luciferase activity. The resultsobtained are reported in FIG. 5.

In the absence of Noggin, co-transfection with HJV cDNA increased BREluciferase activity 10 fold above baseline (compare bar 2 to bar 1).Similarly, incubation with exogenous BMP-2 increased BRE luciferaseactivity 12 fold over baseline (compare bar 4 to bar 1). Thisstimulation by either HJV or exogenous BMP could be blocked by thepresence of Noggin protein (bars 3, 5). In contrast, Noggin did notaffect TGF-β1 induced CAGA luciferase activity (bars 6-8). This datasuggests that HJV generates BMP signals in a ligand-dependent manner,presumably via endogenously expressed BMP ligands.

Result 3: HJV.Fc Binds BMP-2 Selectively

HJV.Fc was incubated overnight with ¹²⁵I-labeled BMP-2 with or withoutexcess cold BMP-2, -4, -7 or TGF-β1, followed by incubation on protein Acoated plates and determination of radioactivity (FIG. 6(A) and FIG. 7).Alternatively, chemical crosslinking of HJV.Fc with ¹²⁵I-labeled BMP-2was performed using DSS in a cell free system (FIG. 6(B)).

As shown in FIG. 6(A) HJV.Fc was able to bind to ¹²⁵I-BMP-2 in a dosedependent fashion. Binding of HJV.Fc to¹²⁵I-BMP-2 was competitivelyinhibited by excess cold BMP-2 but not by BMP-4, BMP-7 or TGF-β1 (seeFIG. 7). ¹²⁵I-BMP-2 can be chemically crosslinked with HJV.Fc in thepresence of DSS (FIG. 6(B), lane 4) and this can be inhibited by excesscold BMP-2 (lane 5). As negative controls, no band was seen in theabsence of DSS (lanes 1 and 2) or when buffer alone (lane 3) or ALK5.Fc(a TGF-β type I receptor, lane 6) was used in place of HJV.Fc.

Result 4: HJV-Mediated BMP Signaling is Inhibited by Dominant NegativeType I Receptors ALK3 and ALK6 and by Dominant Negative Smad1

HepG2 cells were co-transfected with BRE-Luc and HJV either alone or incombination with dominant negative BMP type I receptor ALK3 (ALK3 DN) orALK6 (ALK6 DN) (FIG. 8(A)), or with wildtype (WT) versus dominantnegative (DN) R-Smad 1 (FIG. 8(B)). Transfected cells were thenincubated in the presence or absence of 0.5 nM BMP-2 for 16 hoursfollowed by measurement of luciferase activity.

As shown in FIG. 8(A), transfection with HJV or incubation of cells withexogenous BMP-2 increased BRE luciferase activity above baseline ˜15-20fold (compare bars 2 and 5 to bar 1). This stimulation by either HJV orexogenous BMP-2 could be blocked by co-transfection with dominantnegative ALK3 (bars 3, 6) or dominant negative ALK6 (bars 4, 7).

As shown in FIG. 8(B), transfection with WT Smad 1 alone increased BREluciferase activity ˜12 fold above baseline (compare bars 2 to 1). Incontrast, transfection with DN Smad 1 alone decreased BRE luciferaseactivity below baseline (compare bar 3 to bar 1). This provides furthersupport that there is basal signal transduction via the BMP pathway inthese cells in the absence of exogenously added ligand, and thissignaling can be augmented by the presence of additional WT Smad 1 andinhibited by DN Smad 1. Transfection with HJV increased BRE luciferaseactivity ˜12 fold above baseline (bar 4). Co-transfection of WT Smad 1with HJV further augmented the signaling induced by either WT Smad 1 orHN alone (compare bar 5 to 2, 4). Co-transfection of DN Smad 1 with HNblocked the increase in signal seen with HJV alone (compare bar 6 to bar4). Similar results were seen for the effect of WT Smad 1 and DN Smad 1on exogenous BMP-2 stimulation (bars 7-9). Thus, HJV-mediated BMPsignaling occurs via the classical BMP signaling pathway through BMPtype I receptors ALK3 and ALK6 as well as R-Smad1.

Result 5: Production and Characterization of Mutant HJVG313 V andHJVG3I3V.Fc Fusion Protein

The most common mutation in HJV resulting in juvenile hemochromatosis isa point mutation substituting valine for glycine at amino acid 320(corresponding to amino acid 313 in murine HJV). Mutant HJVG313V andsoluble HJVG313V.Fc cDNA were made using PCR and subcloning techniquesas described above, transfected into CHO cells, and analyzed by reducingSDS PAGE followed by Western blot with anti-HJV antibody (FIGS. 9(A) and(B) left panel) or anti-Fc antibody (FIG. 9(B), right panel).Alternatively, unpermeabilized transfected cells were analyzed byimmunofluorescence microscopy using anti-HJV antibody (FIG. 10).

As shown in FIG. 9(A), mutant HJVG313V is expressed in CHO cells, butmigrates with a different pattern than wild-type HJV suggesting it isprocessed differently, at least in this cell type. Mutant HJVG313V.Fcalso appears to be processed differently from wild-type HN.Fc with aloss of the ˜60 kDa band (see FIG. 9(B)).

As shown in FIG. 10, both wildtype HJV and mutant HJVG313V are expressedon the cell surface in a punctate distribution.

Result 6: Mutant HJVG3I3V Decreases BMP Signaling Ability Compared toWild-Type HJV

HepG2 cells were transfected with BRE-Luc alone or in combination withincreasing concentrations of wildtype HJV or mutant HJVG13V cDNA.Transfected cells were incubated in the presence or absence of 0.5 nMBMP-2 for 16 hours followed by measurement of luciferase activity. Asshown in FIG. 11, in the absence of exogenous ligand, wildtype HJVincreased BRE luciferase activity up to 23 fold over baseline. Thisstimulation was on the order of that seen with 0.5 nM exogenous BMP-2.In contrast, mutant HJVG313V increased BRE luciferase activity only to amaximum of 9 fold. This suggests that mutant HJVG313V, which in humanscan result in juvenile hemochromatosis, has decreased BMP signalingability in liver cells, raising the question of whether BMP signalingmight play a role in iron metabolism.

Conclusions

As reported above, the Applicants have shown that (1) HJV induces BMPbut not TGF-β signaling; (2) HJV signaling is blocked by Noggin, awell-known BMP inhibitor; (3) HJV binds directly to radiolabeled BMP-2ligand; (4) HJV signals via the BMP type I receptors, ALK-3 and ALK-6;(5) HJV signals via the BMP R-Smad, Smad1; (6) an HJV mutant known tocause juvenile hemochromatosis decreases BMP signaling ability; and (7)BMP increases, while Noggin decreases, hepcidin expression in livercells.

These results suggest that HJV is a novel BMP co-receptor whose BMPsignaling ability is important in regulating iron metabolism. Mutationsin HJV could lead to decreased BMP signaling in liver cells, which couldthen decrease hepcidin expression, thereby explaining why persons withHJV mutations have depressed hepcidin levels and thus iron overload. Thepresent findings regarding the novel mechanism of action of HJV reveal aheretofore undiscovered link between BMP signaling and iron metabolism,and could lead to novel treatment strategies of disorders of ironmetabolism such as hemochromatosis and anemia of chronic disease.

Example 3 Effects of BMP-2 on Iron Binding Capacity in Vivo

Study Protocol: Normal mice were injected intraorbitally with 18 μg ofBMP-2 (equivalent to 1 mg per kg body weight), or with carrier solutionas a control. After 4 hours, blood was harvested and serum iron levelsand total iron binding capacity was measured using colorimetric assays.

As shown on FIG. 12, the injection of BMP-2 led to significant decreasesin both the serum iron and the total iron binding capacity. This resultindicates that BMP ligands and BMP inhibitors will be useful astherapeutic agents to regulate iron levels in whole animals includinghumans.

Example 4 Proteolytically Stable HJV, RGMa, and Dragon Mutants

Experiments were undertaken to demonstrate the feasibility of producingmutant HJV, RGMa and Dragon proteins that, in contrast to theircorresponding wild-type proteins, do not undergo proteolytic cleavage.

Mouse RGMa-D169A.Fc mutant cDNA was generated and expressed in HEK cellsupernatants. FIG. 13 shows that the purified protein obtained is notproteolytically cleaved compared to wild-type mouse RMGa.Fc protein.

Similarly, mouse Dragon-D171A.Fc mutant cDNA was generated and expressedin HEK cell supernatants. The purified mutant protein obtained was shownto be stable to proteolytic cleavage compared to wild-type mouseDragon.Fc protein (see FIG. 14).

In a third experiment, the human HJV-D172A mutant cDNA was generated andexpressed in HEK cell supernatants. As shown on FIG. 15, in contrast tothe wild-type human HJV protein, the mutant HJV protein did not undergoproteolytic cleavage.

Mutant HJV, RGMa, and Dragon fusion proteins that are more stable toproteolytic cleavage than the wild-type versions could be advantageouslyused in the methods of the present invention.

Example 5 Mutations in HFE2 Cause Iron Overload in Chromosome 1q-LinkedJuvenile Hemochromatosis

Juvenile hemochromatosis is an early-onset autosomal recessive disorderof iron overload resulting in cardiomyopathy, diabetes and hypogonadismthat presents in the teens and early 20s (refs. 1,2). Juvenilehemochromatosis has previously been linked to the centromeric region ofchromosome 1q (refs. 3-6), a region that is incomplete in the humangenome assembly. Here we report the positional cloning of the locusassociated with juvenile hemochromatosis and the identification of a newgene crucial to iron metabolism. We finely mapped the recombinantinterval in families of Greek descent and identified multipledeleterious mutations in a transcription unit of previously unknownfunction (LOC 148738), now called HFE2, whose protein product we callhemojuvelin. Analysis of Greek, Canadian and French families indicatedthat one mutation, the amino acid substitution G320V, was observed inall three populations and accounted for two-thirds of the mutationsfound. HFE2 transcript expression was restricted to liver, heart andskeletal muscle, similar to that of hepcidin, a key protein implicatedin iron metabolism⁷⁻⁹. Urinary hepcidin levels were depressed inindividuals with juvenile hemochromatosis, suggesting that hemojuvelinis probably not the hepcidin receptor. Rather, HFE2 seems to modulatehepcidin expression.

Two families with juvenile hemochromatosis not linked to 1q wererecently found to have loss-of-function mutations in the gene encodinghepcidin¹⁰. Hepcidin is a small peptide hormone predominantly secretedby the liver¹¹, whose levels correlate inversely with rates of ironuptake in the gut and with the release of iron from macrophages^(12, 13)(FIG. 16). The clinical and biochemical phenotype of 1q-linked juvenilehemochromatosis is indistinguishable from that of hepcidin-deficientjuvenile hemochromatosis, both having intestinal iron hyperabsorptionleading to an early onset of severe iron overload associated withmacrophages that do not load iron. This suggests that the more commonlymutated gene underlying 1q-linked juvenile hemochromatosis gene probablyalso functions in the hepcidin pathway.

To identify the gene associated with 1q-linked juvenile hemochromatosis,we collected samples from 12 unrelated families with juvenilehemochromatosis from Greece, Canada and France, 7 of whom werepreviously reported to be consistent with linkage to the juvenilehemochromatosis locus at 1q21 (HFE2; OMIM 602390). Only one family, JH7,is known to be consanguineous. Parents of all probands, whereascertained, were clinically and biochemically normal.

We verified absence of mutations of hepcidin in all 12 families andconfirmed that juvenile hemochromatosis was consistent with linkage to1q21 in these families by mapping a combination of publicly availablemarkers and 18 new microsatellite markers identified from genomicsequence. Nine of the ten Greek families showed extended markerhomozygosity in the 1q region (FIG. 17), consistent with linkage to acommon gene as the chief determinant of juvenile hemochromatosis in thispopulation. We reconstructed five different Greek haplotypes segregatingin these families, one of which was observed repeatedly. Families JH4,JH8 and JH9 were each homozygous with respect to different haplotypes.The proband in family JH11 segregated alleles consistent withheterozygosity with respect to the common haplotype and a new haplotype.

We carried out multipoint linkage analysis to determine the statisticalsignificance of the observed haplotype sharing and obtained a peakmultipoint lod score of 4.05 in the shared segment for the Greek andCanadian families combined. The April 2003 genome sequence assembly(build 33) contains numerous gaps and duplications, but we were able toestimate the size of the linkage interval and define the linkageboundaries on the basis of existing sequence contigs. Recombinant eventsplaced outer boundaries at CA3AL590452 and CA3AL359207.

We next embarked on a positional cloning effort. According to ourinterpretation of the genome assembly, the region of ˜1.7 Mb associatedwith juvenile hemochromatosis contains 21 RefSeq annotated genes. In thecourse of sequencing these genes, we identified multiple mutations inone particular gene in the minimal recombinant interval (Table 1).

TABLE 1 Genetic and clinical information of families with mutations inHFE2 Number of affected Serum Transferrin individuals Age at Age atferritin saturation Hypo- Arthro- Individual Origin in family onsetdiagnosis (μg I−¹) (%) gonadism pathy JH1-301 Canada 3 7 7 339 94 − −JH3-201 Greece 1 21 25 2,283 100 + + JH4-203 Greece 1 39 49 4,127 90 + +JH5-201 Greece 2 32 39 3,553 100 JH6-205 Greece 2 25 32 2,500 100 + +JH7-201 Greece 3 20 21 NA 100 + − JH8-202 Greece 1 26 33 5,900 98 + −JH9-201 Greece 2 28 33 1,125 80 + + JH10-201 Greece 1 21 25 5,250 100 +− JH11-201 Greece 1 33 37 731 100 − − JH12-201 Greece 1 29 31 2,254100 + − JH13-301 France 1 16 23 7,125 83 + + Effect on Skin GlucoseHeart Hepatic Mutation coding Individual pigmentation intolerancedisease fibrosis status sequence JH1-301 + − − + Compound I222N,heterozygous G320V JH3-201 + − − + Homozygous G320V JH4-203 + − − +Homozygous I281T JH5-201 Homozygous G320V JH6-205 + + + + HomozygousG320V JH7-201 + − − NA Homozygous G320V JH8-202 + − − + HomozygousC361fsX366 JH9-201 − + − + Homozygous G99V JH10-201 − − − + HomozygousG320V JH11-201 + − − − Compound G320V, heterozygous R326X JH12-201 − − −NA Homozygous G320V JH13-301 + + + + Homozygous G320V +, present; −,absent: NA, information not available.

This gene corresponds to anonymous transcript LOC148738 in RefSeq,although we predicted a slightly more complex gene structure fromavailable cDNA and expressed-sequence tag (EST) evidence (FIG. 18 a).The observed mutations include four missense mutations in residues thatare highly conserved in evolution (Table 1 and FIG. 18 b), a prematuretermination mutation and a frameshift mutation. We detected sixdifferent mutations accounting for all 24 alleles in the ten Greekfamilies, one Canadian family and one French family. None of themutations was observed in over 500 control chromosomes. The mutationscosegregated completely with the juvenile hemochromatosis phenotype, andresults of microsatellite-based haplotype analysis were consistent withrecessive inheritance and full penetrance. We observed one commonmutation, the G320V missense variant, in the seven Greek families whoshare the common Greek haplotype and in Canadian and French families.

We predict that hemojuvelin is transcribed from a gene of 4,265 bp intoa full-length transcript with five spliced isoforms (FIG. 18 a). Theputative full-length protein from the longest transcript (transcript 1)is 426 amino acids; the occurrence of this transcript in humans has beenconfirmed experimentally by RT-PCR and sequencing of a novel cDNA clone.Hemojuvelin contains multiple protein motifs (FIG. 18 a) consistent witha function as a membrane-bound receptor or secreted polypeptide hormone.Orthologs of human hemojuvelin are found in mouse, rat and zebrafish(FIG. 18 b). Sequence comparison shows that human hemojuvelin is >85%identical to the mammalian orthologs and ˜45% identical to the fishortholog. The hemojuvelin isoform of 426 amino acids also sharesconsiderable sequence similarity with the repulsive guidance molecule 14(RGM or RGMA) of human (48% identity) and chicken (46% identity; FIG. 18b). In humans there is a third RGM-like protein, RGMB, whose biologicalfunction is currently unknown.

We examined HFE2 expression in 16 human tissue types by probing northernblots with a probe from exon 4 and detected substantial expression inadult and fetal liver, heart and skeletal muscle (FIG. 19). The primaryRNA observed in these tissues migrated at about 2.2 kb, consistent withfull-length transcript 1 in FIG. 18 a. After reprobing the same blotsfor hepcidin, we detected strong expression in adult and fetal liveronly. We later detected expression of hepcidin in a heart-specificnorthern blot.

We measured hepcidin peptide levels in urine samples from a subset ofGreek individuals with juvenile hemochromatosis. Deleterious mutationsof hemojuvelin reduce hepcidin levels despite iron overload, whichnormally induces hepcidin expression¹⁵. Hepcidin levels wereconsistently depressed in the individuals with juvenile hemochromatosis:homozygous affected individuals from five different families had 5-11 ngmg⁻¹ creatinine compared with 14-165 ng mg⁻¹ creatinine in fourheterozygous unaffected carriers and 10-100 ng mg⁻¹ creatinine inunrelated controls. In one individual who did not have hemochromatosiswho had an infection at the time of measurement, urine hepcidin levelwas very high (1,024 ng mg⁻¹ creatinine), as expected. These resultssuggest that HFE2 acts as a modulator of hepcidin expression, althoughit is not possible to distinguish a pretranscriptional from apost-transcriptional or even post-translational role for HFE2 in theabsence of liver biopsies to measure hepcidin mRNA levels. Inadult-onset hereditary hemochromatosis¹⁶ and Hfe knockout mice^(17, 18),hepcidin levels are inappropriately low for the degree of iron overload.Thus, we believe that juvenile hemochromatosis and adult-onsethereditary hemochromatosis are on the same biochemical and phenotypicspectrum, with juvenile hemochromatosis representing the more severe,earlier-onset phenotype with absent (or very low) hepcidin, andadult-onset hemochromatosis manifesting later in life with only partialdeficiency for hepcidin¹⁸ (FIG. 20). The direct result of this hepcidindeficiency is that in both adult-onset hemochromatosis and juvenilehemochromatosis there is intestinal iron hyperabsorption. The excessiveiron uptake in juvenile hemochromatosis is greater than that seen inadult-onset hereditary hemochromatosis, reflecting the lower levels ofhepcidin associated with juvenile hemochromatosis and culminating in anearlier onset of a more acute phenotype.

Loss of function of hepcidin in mice also leads to severe iron overload,mimicking the biochemical and clinical phenotype of juvenilehemochromatosis⁸. In contrast, in both other animal models¹⁹ and humandiseases²⁰, overexpression of hepcidin leads to macrophage ironretention and an iron-deficient phenotype typical of the irondisturbances found in anemia of inflammation (also called anemia ofchronic disease)²¹. Anemia of inflammation is an acquired disorder, seenin individuals with various conditions including infection, malignancyand chronic inflanunation²². It is characterized by a retention of ironby macrophages and decreased intestinal iron absorption, which leads toreduced iron availability for erythropoesis^(21, 23)

Consistent with the proposed role of hepcidin in the pathogenesis ofanemia of inflammation, the defects in iron absorption and reuse inanemia of inflammation are accompanied by elevated urinary (andpresumably serum) hepcidin levels. Existing therapy for anemia ofinflammation is mainly targeted to treating the underlying disorder,with no efficacious treatment specifically directed to amelioration ofthe iron deficiency. Therapeutics that mimic the juvenilehemochromatosis phenotype (hepcidin deficiency) will serve to reducehepcidin levels and thereby treat the opposite phenotype of anemia ofinflammation (hepcidin excess). Thus, hemojuvelin represents a newtherapeutic target for the treatment of anemia of inflammation.

Recent reports that adult-onset hereditary hemochromatosis can resultfrom digenic inheritance with compound heterozygosity with respect toHFE and HAMP (encoding hepcidin) and that mutations in HAMP cancontribute to the severity of adult-onset hereditary hemochromatosis24suggest that modulation of other genes in the hepcidin pathway may alsopredispose to the adult phenotype. Therefore, we are currently exploringthe role of hemojuvelin in modulating the onset and severity ofadult-onset hemochromatosis. The identification of hemojuvelin presentsnew therapeutic and diagnostic opportunities for the management ofiron-related disorders.

Methods

Selection of study subjects. All samples used in this study werecollected with informed consent and approved for study by institutionalreview boards and ethics committees at all affiliated institutions.Families JH3-JH7 were previously reported⁵ as families 1-5,respectively; families JH8 and JH9 were also previously reported⁴ asfamilies 1 and 2, respectively. Diagnoses of affected individuals inthese families were previously reported^(4, 25). We diagnosed additionalprobands with juvenile hemochromatosis based on early presentation withdisease-related clinical complications, including hypogonadotrophichypogonadism, heart disease and skin pigmentation, along with testing oftransferrin saturation, serum ferritin levels and hepatic siderosis(Table 1). Families JH11 and JH13 consist of individual probands.Families JH3, JH4 and JH5 originated in a small area in southwesternGreece; families JH6 and JH7 originated in a mountain area of centralGreece; and family JH10 originated from northwestern and northeasternGreece (maternal and paternal sides, respectively). Families JH8, JH9,JH11 and JH12 also live in Greece, and family JH13 lives in France.Family JH1 lives in Canada and is of European origin. Consanguinity hasbeen documented only in family JH7 (marriage between first cousins).Control DNAs included at least 90 DNAs from each of three differentsources: Greek, northern European and the Coriell polymorphism discoveryresource containing multiple ethnicities.

Markers and genotyping. We used commercially available markers (ABI) forgenotyping in the 1q21 interval. We designed an additional 18 custommarkers using existing sequence obtained from GenBank for fine-mapping.We carried out radiation hybrid mapping on selected microsatellitemarkers or other sequence-tagged sites using the TNG hybrid panel(Research Genetics) to resolve contig order. We carried out genotypingon an Applied Biosystems PRISM® 3100 Genetic Analyzer runningGENEMAPPER® software. We verified mendelian inheritance of alleles forall markers using the PedCheck program26.

Linkage and haplotype analysis. We estimated allele frequencies from 16untransmitted haplotypes from the ten Greek pedigrees and from 40genotyped control Greek individuals. We carried out multipoint linkageanalysis with Genehunter using an inheritance model with 0.99penetrance, 0.000005 phenocopy rate and a recessive disease allelefrequency of 0.01. We determined haplotypes using Genehunter.

Mutation detection. We designed primers to amplify coding sequences forgenes present in the defined 1q interval. The process of primer designinvolved the identification of candidate genes and their respectiveexons in Ensemb1, automated primer design for the exons using Primer3and validation of the primers using e-PCR. We amplified PCR productsusing standard PCR conditions with QIAGEN™ Taq polymerase on a PeltierThermal Cycler (MJ Research, PTC-225). We treated PCR products with 4units exonuclease and 4 units shrimp alkaline phosphatase for 2-16 h andused 5 μl for sequencing. We carried out sequencing using BIGDYE®Terminator on an ABI 3700 sequencer (ABI) and sequence analysis andmutation detection using the Phred/Phrap/Consed/Polyphred^(27,28) orSEQUENCHER® software suites. We designed additional primers to amplifyand sequence all published and predicted exons of LOC148738, includingthe 5′ and 3′ untranslated regions and the 500-bp presumptive sequenceupstream of the first exon. All primer sequences are available onrequest.

Northern-blot analysis. We purchased Clontech northern blots and probedthem with 32P-labeled probes. We generated substrates for probes frompurified, PCR-amplified products from genomic DNA for LOC148738 and HAMPand from manufacturer's supplied reagents for actin according tomanufacturer's instructions. RNA for RT-PCR was either purchased fromClontech or Biochain or prepared from tissues using the QIAGEN™ RNeasyProtect Midi kit. We prepared single-strand cDNA using the INVITROGEN™SUPERSCRIPT® First-Strand Synthesis for RT-PCR kit according to themanufacturer's instructions.

Bioinformatics. We carried out all pairwise sequence comparisons usingBLAST 2 Sequences29 with the following parameters: program, blastp;matrix, BLOSUM62; open gap penalty, 11; extension gap penalty, 1;gap_x_dropoff, 50; word size, 3; expect, 10. We aligned sequences usingClustalX.

We identified orthologs of human hemojuvelin in mouse (although proteincoding potential annotated in the database does not correspond tofull-length open reading frame of the actual sequence), rat andzebrafish (identified by a sequence similarity search of genes predictedby Genscan, gene structure based on genomic sequence traces andsupporting ESTs). We identified paralogs of hemojuvelin in human (RGM orRGMA, RGMB) and chicken (RGM) from Blast comparison to GenBank.

Urinary hepcidin assay. Urinary creatinine concentrations were measuredby UCLA Clinical Laboratories. Cationic peptides were extracted fromurine using CM Macroprep (BIORAD™), eluted with 5% acetic acid,lyophilized and resuspended in 0.01% acetic acid. Urinary hepcidinconcentrations were determined by immunodot assay. Briefly, we analyzedurine extracts equivalent to 0.1-4 mg of creatinine along with 0.6-40 nghepcidin standards on dot blots on IMMOBILON™-P membrane (Millipore). Wedetected hepcidin on the blots using rabbit antibody to human hepcidin¹⁵with goat antibody to rabbit horseradish peroxidase as second antibody.We developed the blots by the chemiluminescent detection method(SuperSignal West Pico Chemiluminescent Substrate, Pierce) andquantified them with the Chemidoc cooled camera running Quantity Onesoftware (BIORAD™). Using this assay, we determined the normal range ofurinary hepcidin to be 10-100 ng per mg creatinine (data not shown).

GenBank accession numbers. Translated portion of HFE2 transcript 1,based on a novel sequenced cDNA clone, AY372521; predicted human HFE2transcripts 1-5, BK001575-BK001578 and BC017926, respectively; predictedzebrafish HFE2 translated portion, BK001579. The mouse HFE2 orthologsequence was inferred with modifications from NM_(—)027126; rat HFE2ortholog was inferred with modifications from AK098165 (annotated ashuman but identified as rat by comparison with genomic sequences);zebrafish HFE2 was inferred with modifications from A1437181 andBG985666.

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Please insert new FIGS. 16-20 after FIG. 15 in the specification.

Other Embodiments

Other embodiments of the invention will be apparent to those skilled inthe art from a consideration of the specification or practice of theinvention disclosed herein. It is intended that the specification andexamples be considered as exemplary only, with the true scope of theinvention being indicated by the following claims.

1-102. (canceled)
 103. A composition comprising a fusion proteincomprising a polypeptide comprising an amino acid sequence 86% identicalto SEQ ID NO:24 fused to Fc, wherein the fusion protein increases bonemorphogenic protein signal in vitro.
 104. The composition of claim 103,wherein the Fc is human Fc.
 105. The composition of claim 103, whereinthe Fc is IgG Fc.
 106. The composition of claim 103, wherein thepolypeptide comprises an amino acid sequence 92% identical to SEQ IDNO:24.
 107. The composition of claim 105, wherein the polypeptidecomprises an amino acid sequence 94% identical to SEQ ID NO:24.
 108. Thecomposition of claim 106, wherein the polypeptide comprises an aminoacid sequence 99% identical to SEQ ID NO:24.
 109. The composition ofclaim 103, wherein the polypeptide comprises an amino acid sequence 99%identical to amino acids 1-400 of SEQ ID NO:24.
 110. The composition ofclaim 109, wherein the polypeptide comprises an amino acid sequence 99%identical to amino acids 35-400 of SEQ ID NO:24.
 111. A pharmaceuticalcomposition comprising the fusion protein of claim 103 and apharmaceutically acceptable carrier.